Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period

Abstract

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc(r)) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc(r) genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc(r) genes was quantified by real-time quantitative PCR. To confirm the Tc(r) gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc(r) genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc(r) genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.

FIG. 1.

Locations of monitoring wells and direction of groundwater flow at sites A and C. Stratigraphic columns indicate the locations and characteristics of sand layers. Large open arrows indicate the direction of groundwater flow. The locations of monitoring wells are indicated by circles; open circles at site A represent nested wells screened in the deeper sand layer. Numbers in parentheses are well depths, in meters. The black rectangles represent confinement buildings.

FIG. 2.

Total detection frequency (a and c) and proportion (b and d) of seven tetracycline resistance genes at two swine facilities between 2000 and 2003 determined by PCR. Total detection frequency was calculated by summing the number of positive detections of all seven tet genes found in the lagoon and groundwater samples at each sampling time. The proportion of detection frequency for each tet gene was expressed as percentage of detection frequency for each gene against total detection frequency over the 3-year monitoring period. Sample number (n) was calculated as average sample number (16.0 for site A and 6.6 for site C) multiplied by 7 (the number of targeted tet genes).

FIG. 3.

PCA biplot of tetracycline resistance gene occurrence in groundwater wells and lagoon samples. Plotted points represent the centroids of the data from six sampling dates for each well, and error bars show the bivariate variance. Vectors display the loadings of tet gene presence and absence in wells on the first two principal component axes. Triangles represent wells from site A (white, background wells; light gray and upward pointing, wells in shallow sand layer; dark gray and downward pointing, wells in deep sand layer), circles represent wells from site C, and squares represent lagoon samples. For well locations, see Fig. 1.

FIG. 4.

BI dendrogram showing the phylogenetic affiliations of tet(W) sequences from lagoon and groundwater samples. For the sequences from the waste samples, clone names begin with “WS” followed by sample source (AP, manure solids in concrete settling basin at site A; AL, waste in lagoon at site A; CL, waste in lagoon at site C) and clone number. For the sequences from groundwater samples, clones are named with the prefix “GW” followed by well number and clone number. Sequences from manure samples are shown in boldface. The number at the node indicates the probability of branching obtained by 10 million generations; 1.00 corresponds to 100% support of the branching. Bar, nucleotide substitutions per sequence position. Sequence groups IG-1 to IG-5 are boxed within the tree. Within each IG group, all clones had identical nucleotide sequences. The numbers in parentheses for IG groups indicate the number and source site of sequences belonging to each group.