The global spread of antibiotic resistance genes (ARGs) has resulted in significant societal and economic costs of treating antibiotic-resistant bacterial infections. Even countries that have made substantial efforts to decrease the misuse and overuse of antibiotics still experience increasing rates of clinical antibiotic resistance, emphasizing the complexity of this problem. This dissertation contributes to improving the understanding of overlooked ARG sources and dissemination pathways, as well as the development of ARG control strategies to enhance the mitigation of the associated risks.
Wildlife carrying resistome, especially wild birds, could be an important but overlooked ARG reservoir and dissemination vector, and urbanization could amplify such risks as it fosters many overlapping habitats and frequent interactions between wildlife populations and human communities. We analyzed fresh feces from three common bird species living in urban environments (Houston metropolitan areas). ARGs encoding resistance to three major classes of antibiotics (i.e., tetracyclines, β-lactams, and sulfonamides) and the mobile genetic element integrase gene (intI1) were abundant (up to 1e+10 copies/g dry feces), with relative concentrations surprisingly comparable to that in poultry and livestock that are occasionally fed with antibiotics. Biomarkers for opportunistic pathogens were also abundant (up to 1e+7 copies/g dry feces) and the dominant isolates (i.e., Enterococcus spp. and Pseudomonas aeruginosa) harbored both ARGs and virulence genes. ARGs and intI1 in these bird feces exhibited moderate persistence and increased the local resistome in the receiving soil, indicating an enlarged region of influence beyond the bird feces. Horizontal gene transfer (HGT) greatly prompts the dissemination of antibiotic resistance into pathogens, and conjugation is often considered to be the major HGT pathway. The contribution of phage-mediated transduction (relative to via conjugation) in various environmental settings remains poorly understood, despite the abundance of phage-borne ARGs. We investigated the influence of bacterial concentration and water turbulence level (quantified as Reynold’s number (Re)) in suspended growth systems on the frequency of ARG transfer by two mechanisms: delivery by a lysogenic phage λ and conjugation mediated by a self-transmissible plasmid RP4. Using Escherichia coli as recipient, phage delivery had a comparable frequency to that of conjugation in suspensions with low bacterial concentration (1e+4 CFU/mL) and moderate turbulence (Re = 5e+4). At 1e+7 CFU/mL, no significant difference was observed between the frequencies of ARG transfer by the two mechanisms under quiescent water conditions or when Re reached 5e+5, corroborating the simulation of cell- (or phage)-to-cell collisions. The frequency of ARG dissemination via phage delivery might be further enhanced due to the co-occurrence with aged microplastics, which can act as sinks of common chemical contaminants (e.g., antibiotics) and microbes. Here, we used UV-aged polystyrene microplastics (PS-MPs) to investigate how microplastic aging affects phage delivering ARGs. Relative to pristine PS-MPs (MP0), the adsorption capacity of MP20 (20-day UV-aged PS-MPs) towards phage λ and recipient cell E. coli increased by 8.3- and 6.6-fold, respectively. Moreover, MP20 released more organic compounds (possibly depolymerization byproducts) that upregulated phage infection associated genes. Accordingly, MP20 enhanced the frequency of phage-mediated ARG transfer by 3.5-fold. Lysogenic phage might facilitate ARG dissemination, while polyvalent lytic phage can be harnessed for efficient killing of antibiotic resistant pathogens. Biofilms pervasive in drinking water systems can harbor resistant pathogens and thus raise great health concerns. Here, we explored the use of peptide display on engineered filamentous coliphage M13 for precise delivery of polyvalent lytic phages to control resistant pathogens in biofilms. M13 phage was dual-modified to display biofilm-binding peptides (presenting high affinity to P. aeruginosa polysaccharides) on major coat protein (pVIII) and peptides specific for polyvalent lytic phage on tail fiber protein (pIII). The modified M13 had 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than unconjugated polyvalent lytic phage. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilms and were more effective than polyvalent lytic phages alone in reducing live bacterial biomass (84% vs 34%). Biofilm regrowth was also mitigated as phage conjugates further downregulated bacterial genes associated with biofilm formation. Overall, these findings characterize some overlooked ARG reservoirs and dissemination vectors that require mitigations. Wild birds are important carriers of ARGs in urban areas. The contribution of phages to ARG horizontal transfer is sometimes comparable to that of conjugation, and it can be considerably enhanced in the presence of aged microplastics. Phages can also target resistant pathogens harbored in biofilms, and their delivery could be enhanced by peptide display strategies that enable biofilm eradication in drinking water storage and distribution systems.