Effect of tetracycline on transfer and establishment of the tetracycline-inducible conjugative transposon Tn916 in the guts of gnotobiotic rats

Abstract

We have investigated the transfer of Tn916 among strains of Enterococcus faecalis OG1 colonizing in the intestines of gnotobiotic rats. This animal model allows a low limit of detection and efficient colonization of the chosen bacteria. The animals continuously received tetracycline in drinking water. A tetracycline-sensitive recipient strain was allowed to colonize the animals before the resistant donor was introduced. The numbers of donors, recipients, and transconjugants in fecal samples and intestinal segments were estimated. The bioavailable amounts of tetracycline in fecal samples and intestinal segments were monitored by using bacterial biosensors carrying a transcriptional fusion of a tetracycline-regulated promoter and a lacZ reporter gene. Chromosomal locations of Tn916 in transconjugants isolated either from the same animal or from different animals were compared by Southern blot analysis. Our results indicated that selection for the resistant phenotype was the major factor causing higher numbers of transconjugants in the presence of tetracycline. Tetracycline-sensitive E. faecalis cells colonized the intestine even when the concentrations of tetracycline in feces and intestinal luminal contents exceeded growth-inhibitory concentrations. This suggests the existence of tetracycline-depleted microhabitats in the intestinal environment.

FIG. 1.

CFU of recipient E. faecalis OG1RF (open squares), donor E. faecalis OG1SS::Tn916 (closed squares), and transconjugant E. faecalis OG1RF::Tn916 (triangles) in fecal samples from rats receiving tetracycline at either 0.0 μg/ml (a), 5 μg/ml (b), 10 μg/ml (c), or 50 μg/ml (d) in drinking water. At days 0 and 7, respectively, the recipient and the donor strains were introduced. Each point represents the geometric average of values obtained from three animals. Error bars indicate standard errors of the means.

FIG. 2.

Southern blots of HindIII-digested transconjugant DNAs isolated from the same animal (a) and from eight different animals (b). The fragment lengths are between 7 and 14 kb in all lanes. Lanes 1 to 8, transconjugants isolated from the same animal in group B. Lanes 9 and 10, transconjugants from group D. Lanes 11 and 12, transconjugants from group C. Lanes 13 and 14, transconjugants from group B. Lanes 15 and 16, transconjugants from group A. Lane 17, DNA from recipient OG1RF. Lane 18, DNA from donor OG1SS::Tn916. The DNAs in lanes 7 and 14 originate from the same transconjugant.

FIG. 3.

Doubling times (generation times) of the tetracycline-sensitive recipient E. faecalis OG1RF obtained with different tetracycline concentrations during balanced growth in LB broth. Each point represents the average of three values. Error bars indicate standard errors of the means.