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

Abstract

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.

FIG. 1

Maps of sites A and C and corresponding stratigraphic columns indicating the locations and characteristics of sand layers. The direction of groundwater flow is indicated by large open arrows, and the locations of monitoring wells are indicated by circles; open circles represent nested wells screened in deeper sand layers. Well depths (in meters) are indicated in parentheses. The solid rectangles represent confinement buildings.

FIG. 2

DGGE analysis of tet(M) in water samples and in bacterial isolates. Lane 1, site A lagoon; lane 2, A8; lane 3, A9; lane 4, A16; lane 5, A3; lane 6, A13; lane 7, A15; lane 8, A5; lane 9, A11; lane 10, A7 (site A background well); lane 11, ALE1; lane 12, ALE2; lane 13, ALE3; lane 14, ALE4; lane 15, A8-2; lane 16, A8-3; lane 17, A8-4; lane 18, AL-2; lane 19, AL-3; lane 20, AL-4; lane 21, A16-2; lane 22, AL-1; lane 23, site C lagoon; lane 24, C2; lane 25, C6; lane 26, C7; lane 27, C1 (site C background well); lane 28, CLE1; lane 29, CLE2; lane 30, CLE3; lane 31, CLE4, lane 32, CL-1; lane 33, CL-3; lane 34, C2-1; lane 35, tet(M) positive control strain; lane 36, CL-2. Lanes M contained markers consisting of tet(Q), tet(M), and tet(O).

FIG. 3

DGGE analysis of V3 variable region of 16S rDNA from tetracycline-resistant bacterial isolates. Lane 1, ALE1; lane 2, ALE2; lane 3, ALE3; lane 4, ALE4; lane 5, A8-2; lane 6, A8-3; lane 7, A8-4; lane 8, AL-2; lane 9, AL-3; lane 10, AL-4; lane 11, A16-1 (fungal isolate, negative control); lane 12, AL-1; lane 13, CLE1; lane 14, CLE2; lane 15, CLE3; lane 16, CLE4; lane 17, CL-1; lane 18, CL-3; lane 19, CL-2.

FIG. 4

Phylogenetic placement of tetracycline resistance genes encoding RPPs. The sequence of the Aquifex aeolicus fusA gene encoding translation elongation factor EF-G is used as the outgroup for rooting the tree. The numbers above nodes indicate the number of times that a tree configuration occurred among 1,000 bootstrap trials. Scale bar = 0.1 fixed nucleotide substitution per sequence position. The tet(M)-harboring strains isolated in this work are indicated by boldface type. These strains were not incorporated into the phylogenetic analysis and were placed in the Tet M cluster arbitrarily based on sequence similarity.