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

Abstract

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.

FIG. 1.

Maximum-likelihood tree of tetracycline resistance genes, encoding tet efflux pumps of gram-negative bacteria (subcluster tet), and other genes of the DHA12 family. The same overall topology was obtained by neighbor-joining and parsimony analyses. Numbers at nodes represent the percentages of occurrence of nodes in 100 bootstrap trials. The scale bar is in fixed nucleotide substitutions per sequence position. The tree is arbitrarily rooted with the P. aeruginosa cmlA gene. The set of PCR primers, presented in Table 2, targets various classes of tet genes, which are shown in boldface.

FIG. 2.

Maps of sites A and C and corresponding stratigraphic columns indicating the location and characteristics of sand layers. Large open arrows indicate groundwater flow. Circles indicate the locations of monitoring wells. Open circles represent nested wells screened in deeper sand layers. Well depths in meters are in parentheses. Black rectangles represent confinement buildings.