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. 2012 Jan 18:3:2.
doi: 10.3389/fmicb.2012.00002. eCollection 2012.

IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes

Holger Heuer  1 , Chu T T Binh, Sven Jechalke, Christoph Kopmann, Ute Zimmerling, Ellen Krögerrecklenfort, Thomas Ledger, Bernardo González, Eva Top, Kornelia Smalla
Affiliations

IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes

Holger Heuer et al. Front Microbiol. .

Abstract

The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

Keywords: IncP-1ε plasmid; arable soil; complete sequence; exogenous isolation; gene cassette; pig manure; qPCR.

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Figures

Figure 1
Figure 1
Copy numbers of trfA (replication initiation gene) of IncP-1ε plasmids relative to 16S rRNA gene copies (rrn) in manure and in soil, which was treated either with the antibiotic compound sulfadiazine (SDZ), with manure without antibiotics, with manure containing SDZ, or with water (untreated). Sampling of the soil microcosms was repeated three times. Error bars indicate SD (n = 4). Differing letters show a significant effect of the treatment (ANOVA with repeated measures).
Figure 2
Figure 2
The common plasmid backbone of the completely sequenced IncP-1ε plasmids pHH128, pKJK5, pKS77, pHH3414, and pHH3408. The two insertion sites of accessory elements within the gene parA are indicated.
Figure 3
Figure 3
Accessory regions of the completely sequenced IncP-1ε plasmids pHH128, pKJK5, pKS77, pHH3414, and pHH3408. Homologous regions are indicated by framed areas. Inverted repeats of the transposable elements are indicated by rectangles, target site duplications (direct repeats) are indicated by closed ovals.
See this image and copyright information in PMC

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