Exposure to environmental level pesticides stimulates and diversifies evolution in Escherichia coli towards greater antibiotic resistance

Abstract

Antibiotic resistance is one of the most challenging issues in public health. Antibiotic resistance can be selected by antibiotics at sub-inhibitory concentrations, the concentrations typically occurring in natural and engineered environments. Meanwhile, many other emerging organic contaminants such as pesticides are frequently co-occurring with antibiotics in agriculture-related environments and municipal wastewater treatment plants. To investigate the effects of the co-existing, non-antibiotic pesticides on the development of antibiotic resistance, we conducted long-term exposure experiments using a model Escherichia coli strain. The results revealed that 1) the exposure to a high level (in mg/L) of pesticides alone led to the emergence of mutants with significantly higher resistance to streptomycin; 2) the exposure to an environmental level (in {micro}g/L) of pesticides together with a sub-inhibitory level (in sub mg/L) of ampicillin synergistically stimulated the selection of ampicillin resistance and the cross-selection of resistance to three other antibiotics (i.e., ciprofloxacin, chloramphenicol, and tetracycline). Resistance levels of mutants selected from co-exposure were significantly higher than those of mutants selected from ampicillin exposure only. The comparative genomic and transcriptomic analyses indicate that distinct and diversified genetic mutations in ampicillin- and ciprofloxacin-resistant mutants were selected from co-exposure, which likely caused holistic transcriptional regulation and the increased antibiotic resistance. Together, the findings provide valuable fundamental insights into the development of antibiotic resistance under environmentally relevant conditions, as well as the underlying molecular mechanisms of the elevated antibiotic resistance induced by the exposure to pesticides.\n\nSignificance statementAntibiotic resistance is a major threat to public health globally. Besides clinically relevant environments, the emergence and spread of resistant bacteria in non-clinical environments can also potentially pose risks of therapy failures. This study showed that the long-term, environment-level exposure to pesticides with and without antibiotics significantly stimulated the development of greater antibiotic resistance. The resistant strains selected from the exposure to pesticides are genetically and metabolically distinct from the ones selected by the antibiotic only. Although it is still being debated regarding whether or not a large use of antibiotics in plant agriculture is harmful, our findings provide the first fundamental evidence that greater concerns of antibiotic resistance may result if antibiotics are applied together with non-antibiotic pesticides.