Detection of a common and persistent tet(L)-carrying plasmid in chicken-waste-impacted farm soil

Abstract

The connection between farm-generated animal waste and the dissemination of antibiotic resistance in soil microbial communities, via mobile genetic elements, remains obscure. In this study, electromagnetic induction (EMI) surveying of a broiler chicken farm assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, a disparity existed between the two sites with regard to soil pH, tetracycline resistance (Tc(r)) levels among culturable soil bacteria, and the incidence and prevalence of several tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a relatively pristine regional forest. When the farm was in operation, tet(L), tet(M), tet(O), erm(A), erm(B), and erm(C) genes were detected in the waste-affected soil. Two years after all waste was removed from the farm, tet(L), tet(M), tet(O), and erm(C) genes were still detected. The abundances of tet(L), tet(O), and erm(B) were measured using quantitative PCR, and the copy numbers of each were normalized to eubacterial 16S rRNA gene copy numbers. tet(L) was the most prevalent gene, whereas tet(O) was the most persistent, although all declined over the 2-year period. A mobilizable plasmid carrying tet(L) was identified in seven of 14 Tc(r) soil isolates. The plasmid's hosts were identified as species of Bhargavaea, Sporosarcina, and Bacillus. The plasmid's mobilization (mob) gene was quantified to estimate its prevalence in the soil, and the ratio of tet(L) to mob was shown to have changed from 34:1 to 1:1 over the 2-year sampling period.

Fig 1

Apparent electrical conductivity map of the broiler chicken farm study site. EC_a data (collected from depths of up to 1.5 m) are in the vertical dipole orientation. Buildings are shown in gray. Red stars denote sampling sites.

Fig 2

Prevalence of specific ARGs at the waste-impacted site during routine poultry litter deposition (2008) and 2 years after all waste was removed (2010). The prevalences of a group of broad-host-range mobilizable plasmids that carry tet(L) were estimated by quantifying their mobilization gene, mob. Error bars represent the standard deviations of the results from at least three independent qPCR runs. Numbers in parentheses reflect fold changes in the relative abundances of the genes from 2008 to 2010.

Fig 3

Neighbor-joining tree of TetL efflux pumps. The bootstrap consensus tree was inferred from 1,000 replicates. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates were collapsed. The bar represents 0.02 amino acid substitutions per site. GenBank accession numbers, plasmid names, and host genera are listed. TetL sequences identified in this study are denoted by a star symbol and are shown collectively as being on pSU1.