Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs

Abstract

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10(6) and 10(7) CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10(6) CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10(3) and 10(4) CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.

FIG. 1.

Concentration in fecal samples of total enterococci (open circles), streptomycin-resistant enterococci (closed circles), and E. coli (+). From day 22 of the experiment, the animals were treated with streptomycin. At days 23 and 45, respectively, the recipient and donor strains of E. faecalis OG1 were introduced. The figure shows the average values from three mini-pigs. Error bars represent standard errors of the means.

FIG. 2.

Concentration in fecal samples of recipient E. faecalis OG1SS (open squares), donor E. faecalis OG1RFS(pCF10) (closed squares), and transconjugant E. faecalis OG1SS(pCF10) (closed triangles). At days 23 and 45, respectively, the recipient and donor strains were introduced. The figure shows the average values from three mini-pigs. Error bars represent standard errors of the means.

FIG. 3.

Concentration in various intestinal segments of total aerobic microorganisms (closed diamonds), total anaerobic microorganisms (open diamonds), total tetracycline-resistant aerobic microorganisms (×), E. coli (+), total enterococci (open circles), recipient bacteria (open squares), and transconjugant bacteria (closed triangles). The figure shows the average values from three mini-pigs, measured at the day of euthanatization. Error bars represent standard errors of the means.