Identification of Tet45, a tetracycline efflux pump, from a poultry-litter-exposed soil isolate and persistence of tet(45) in the soil.

ABSTRACT

OBJECTIVES: To characterize a tetracycline resistance (Tc(R)) determinant, closely related to the TetL efflux pump, in a Bhargavaea cecembensis strain previously isolated from a poultry-litter-impacted soil. METHODS: Genomic DNA of B. cecembensis DMV42A was shotgun cloned and expressed in Escherichia coli. Antimicrobial susceptibility testing and a [(3)H]tetracycline uptake assay were used to confirm the function of the target gene. Transferability of the gene was examined using filter matings and confirmed by PCR and sequencing. Real-time quantitative PCR was performed on soil metagenomic DNA to evaluate the prevalence of the gene in the soil from which B. cecembensis DMV42A was isolated and in more pristine local soils. RESULTS: The Tc(R) determinant from B. cecembensis DMV42A, designated Tet45, was identified as a tetracycline efflux pump sharing 78% amino acid identity with certain TetL proteins. In B. cecembensis DMV42A, tet(45) was adjacent to truncated and non-functional arsenic resistance genes with high sequence similarities to genes from staphylococcal plasmids. After filter matings, the tet(45) gene could be found in E. coli transconjugants, although the transfer mechanism was unknown. Tet45 homologues are also present in the genomes of several Bacillus cereus strains and a Bacillus thuringiensis strain. tet(45) was detected in the poultry-litter-impacted soil, and persisted at a similar level 2 years after removal of the chicken waste, although it was not detected in several more pristine soils. CONCLUSIONS: Tet45 is a tetracycline efflux pump closely related to TetL. Horizontal gene transfer may have contributed to the dissemination and persistence of tet(45) in a poultry-litter-impacted soil.