1Antibiotic resistance is one of the most challenging issues in public health. Antibiotic resistance 2 can be selected by antibiotics at sub-inhibitory concentrations, the concentrations typically 3 occurring in natural and engineered environments. Meanwhile, many other emerging organic 4 contaminants such as pesticides are frequently co-occurring with antibiotics in agriculture-related 5 environments and municipal wastewater treatment plants. To investigate the effects of the co-6 existing, non-antibiotic pesticides on the development of antibiotic resistance, we conducted 7 long-term exposure experiments using a model Escherichia coli strain. The results revealed that 8 1) the exposure to a high level (in mg/L) of pesticides alone led to the emergence of mutants with 9 significantly higher resistance to streptomycin; 2) the exposure to an environmental level (in 10 µg/L) of pesticides together with a sub-inhibitory level (in sub mg/L) of ampicillin 11 synergistically stimulated the selection of ampicillin resistance and the cross-selection of 12 resistance to three other antibiotics (i.e., ciprofloxacin, chloramphenicol, and tetracycline). 13 Resistance levels of mutants selected from co-exposure were significantly higher than those of 14 mutants selected from ampicillin exposure only. The comparative genomic and transcriptomic 15 analyses indicate that distinct and diversified genetic mutations in ampicillin-and ciprofloxacin-16 resistant mutants were selected from co-exposure, which likely caused holistic transcriptional 17 regulation and the increased antibiotic resistance. Together, the findings provide valuable 18 fundamental insights into the development of antibiotic resistance under environmentally 19 relevant conditions, as well as the underlying molecular mechanisms of the elevated antibiotic 20 resistance induced by the exposure to pesticides. 21 Escherichia coli 23 3 Significance statement 24 Antibiotic resistance is a major threat to public health globally. Besides clinically relevant 25 environments, the emergence and spread of resistant bacteria in non-clinical environments can 26 also potentially pose risks of therapy failures. This study showed that the long-term, 27 environment-level exposure to pesticides with and without antibiotics significantly stimulated the 28 development of greater antibiotic resistance. The resistant strains selected from the exposure to 29 pesticides are genetically and metabolically distinct from the ones selected by the antibiotic only. 30 Although it is still being debated regarding whether or not a large use of antibiotics in plant 31 agriculture is harmful, our findings provide the first fundamental evidence that greater concerns 32 of antibiotic resistance may result if antibiotics are applied together with non-antibiotic 33 pesticides. 34 35Antibiotic resistance has been one of the most challenging environmental and public health 36 issues. The de novo mutation is one important route for bacteria to acquire antibiotic resistance, 37 under both clinical an...