Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G plus C content

Bioactive amounts of antibiotics as well as resistant bacteria reach the soil through manure fertilization. We investigated plasmids that may stimulate the environmental spread and interspecies transfer of antibiotic resistance. After treatment of two soils with manure, either with or without the sulfonamide antibiotic sulfadiazine, a significant increase in copies of the sulfonamide resistance gene sul2 was detected by qPCR. All sul2 carrying plasmids, captured in Escherichia coli from soil, belonged to a novel class of self-transferable replicons. Manuring and sulfadiazine significantly increased the abundance of this replicon type in a chemically fertilized but not in an annually manured soil, as determined by qPCR targeting a transfer gene. Restriction patterns and antibiograms showed a considerable diversity within this novel plasmid group. Analysis of three complete plasmid sequences revealed a conserved 30 kbp backbone with only 36% G+C content, comprised of transfer and maintenance genes with moderate homology to plasmid pIPO2 and a replication module (rep and oriV) of other descent. The plasmids differed in composition of the 27.0-28.3 kbp accessory region, each of which carried ISCR2 and several resistance genes. Acinetobacter spp. was identified as a potential host of such LowGC-type plasmids in manure and soil.