Unravelling the roles of emerging environmental contaminants in promoting the spread of antibiotic resistance

Abstract

Antibiotic resistance (AR) poses a global threat to public health. It has been well recognized that antibiotics could stimulate the dissemination of AR through mutation and horizontal gene transfer (HGT). However, little is known about whether non-antibiotic environmental contaminants (NAECs) could promote the acquisition of AR. In order to assess whether and how NAECs promote the emergence and spread of AR, this thesis investigated the effects of two categories of representative inorganic and organic NAECs, heavy metals-based nanoparticles (NPs) and the corresponding ions, and triclosan (TCS), on the acquisition of AR through i) mutation, ii) conjugation of antibiotic resistance genes (ARGs) and iii) transformation of ARGs. For the mutation study, the mutagenic potential of one typical NAEC, TCS, was investigated by a 30-day evolution experiment using Escherichia coli K12 MG1655 as a model bacterium. The results indicated that TCS at an environmentally relevant concentration (0.2 mg/L) could significantly promote the development of hereditable multidrug-resistant bacteria through mutagenesis. The combination of whole genome DNA and RNA sequencing analyses with the physiological profiles revealed that TCS could induce over-production Reactive Oxygen Species (ROS), which might result in the genetic mutations in global and local multi-drug resistance regulator and beta-lactamase promoter genes (e.g., fabI, frdD, marR, acrR and soxR). Consequently, the mutations in those genes significantly increased the transcription of beta-lactamase and multi-drug efflux pumps-encoding genes, and repressed the expression of membrane permeability-related genes, thus enhanced the resistance of the mutants against multiple antibiotics. For the conjugation study, two conjugation models were established by using E. coli LE392 carrying RP4 plasmid as the donor, while E. coli K12 MG1655 and Pseudomonas putida KT2440 as intra- and inter-genera recipients, respectively. The effects of AgNPs/Ag+ (the typical heavy metal-based NPs/ion) and TCS on the conjugative transfer of RP4 plasmid were evaluated. Both AgNPs/Ag+ and TCS were found to promote the conjugative transfer of plasmid RP4 between bacterial genera at environmental concentrations. Molecular investigation indicated the AgNPs/Ag+ and TCS-promoted conjugation was associated with the ROS over-production, increased the cell membrane permeability, induced SOS response and enhanced ATP synthesis. More importantly, the enhancement of ROS generation and conjugation frequency-mediated by AgNPs/Ag+ and TCS could be simultaneously repressed by the addition of a ROS scavenger, which confirmed the contribution of ROS over-production on the AgNPs/Ag+ and TCS-promoted conjugation. The possible effects of multiple heavy metal-based NPs and ions (including AgNPs, CuO NPs, ZnO NPs, Ag+, Cu2+, and Zn2+) and TCS on stimulating the natural transformation of ARGs was assessed by using plasmid pUC19 or pWH1266 as gene transfer agent of ARGs, and E. coli DH5ɑ or Acinetobacter baylyi ADP1 as the recipient. The outcomes of the investigations demonstrated all tested heavy metal-based NPs/ions and TCS could promote the bacterial ARGs uptake ability under environmental concentrations. The TCS and Ag/CuO NPs/ions-promoted transformation frequencies were associated with ROS over-production. Furthermore, TCS could provoke the E. coli type IV competence and general secretion system, thereby promoting the plasmid uptake. Additionally, transmission electron microscope imaging revealed the roughened cell membrane after Ag NPs, CuO NPs, Ag+ and Cu2+ exposure. ZnO NPs and Zn2+ might increase the natural transformation rate by stimulating the stress response and ATP synthesis. All tested NPs and ions resulted in up-regulating the competence and SOS response-associated genes. In conclusion, this thesis demonstrated that two types of NAECs, heavy metal-based NPs/ions and TCS at environmental-relevant concentrations, could promote the bacterial acquisition of AR by inducing mutation, stimulating ARGs conjugation and promoting ARGs transformation. By combining the whole-genome DNA and RNA sequencing, proteomics analyses with the selected measurements of physiological profiles, the potential mechanisms associated with the heavy metal-based NPs/ions or TCS-facilitated emergence and dissemination of AR were revealed. These mechanisms include i) ROS over-production, ii) enhancement of cell membrane permeability, iii) promotion of bacterial SOS response and stress response levels, and iv) stimulation of bacterial competence/secretion systems. The findings of this thesis could advance the current understanding of the emergence and acquirement of AR in environmental microbes mediated by NAECs.