Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria.

Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H(+) antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.

Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities.

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.