Diverse aadA gene cassettes on class 1 integrons introduced into soil via spread manure

Class 1 integrons are assumed to play an important role in dissemination of antibiotic resistance genes. Class 1 integrons from clinical isolates often have 5′- and 3′-conserved segments flanking the variable region where gene cassettes integrate. Primers targeting the conserved segments were used to amplify the variable region from total community DNA of manure and soil. Integron gene cassette diversity in manure and manured soils was studied by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. The analyzed group of integrons could be detected in manured soils but not in soil without a history of manure treatment. aadA gene cassettes encoding resistance to streptomycin and spectinomycin were frequently detected in the clone libraries. To elucidate whether “clinical” class 1 integron gene cassettes are typically introduced via manure into agricultural soil, a survey with a unique set of manure samples originating from 15 different pig-producing facilities was performed. aadA gene cassettes were detected in all field-scale manures, confirming that aadA genes localized on class 1 integrons are introduced via piggery manure into agricultural soils. In addition, class 1 integrons without gene cassettes were detected in total community DNA of many manures.