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

Abstract

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.

Specific Microbial Attachment to Root Knot Nematodes in Suppressive Soil

“…PCR with the universal bacterial primers F27/R1494 was performed as previously described (19). The products were purified with a Minelute PCR purification kit (Qiagen, Hilden, Germany) and used as target to amplify the V3-V4 region of 16S rRNA genes with fusion primers containing the Roche-454 A and B Titanium sequencing adapters, an eight-base barcode sequence in adaptor A, and specific sequences V3F/V4R targeting the ribosomal region.…”

Accumulation of Sulfonamide Resistance Genes in Arable Soils Due to Repeated Application of Manure Containing Sulfadiazine

“…Obviously, this was not so much caused by the addition of sul genes originating from manure (S0 treatment) but rather by the selective pressure exerted by bioavailable SDZ in soil. Manure typically contains substantial numbers of resistant bacteria, with 10 Ϫ3 to 10 Ϫ2 sul1 copies and 10 Ϫ2 to 10 Ϫ1 sul2 copies per 16S rRNA gene copy (1,14,15). These numbers were high enough to result in a temporary increase in sul genes in soil after manure application in earlier studies (14,15).…”

“…Manure typically contains substantial numbers of resistant bacteria, with 10 Ϫ3 to 10 Ϫ2 sul1 copies and 10 Ϫ2 to 10 Ϫ1 sul2 copies per 16S rRNA gene copy (1,14,15). These numbers were high enough to result in a temporary increase in sul genes in soil after manure application in earlier studies (14,15). The substrate from manure allows for the growth of populations carrying sul genes under selective pressure of SDZ.…”

“…A survey of 15 field-scale piggery manures showed that piggery manure represents a hot spot for antibiotic resistance genes as well as broad-host-range plasmids (2)(3)(4). In a soil microcosm experiment, significant effects of manure containing SDZ on the abundance of the sulfonamide resistance genes sul1 and sul2 were detected even 2 months after application, compared to the abundance in untreated soil or soil treated with manure devoid of antibiotics (14,15). Also, the frequency of capturing mobile genetic elements conferring SDZ resistance was increased by manure containing SDZ.…”

“…Total community DNA was extracted using the FastPrep FP120 bead-beating system for cell lysis and the FastDNA spin kit for soil (Q-Biogene, Carlsbad, CA). The copy numbers of sul1, sul2, and 16S rRNA genes were quantified by 5Ј-nuclease assays using quantitative real-time PCR as previously described (14,15). Copy numbers of sul genes were related to 16S rRNA gene copy numbers and log transformed (Fig.…”