Enterococcus faecalis gene transfer under natural conditions in municipal sewage water treatment plants

Abstract

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10(5)-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10(5) to 10(8) events per 4 h, indicating that gene transfer should take place under natural conditions.

FIG. 1

Schematic diagram of the microcosm used for experiments in the activated sludge digestors. The whole assembly could be varied in length, by using nuts and bolts, from 15 to 45 cm and was sterilized with the dialysis tubing connected to the polypropylene end caps via autoclavable O rings. The arrows indicate connections via silicone tubing to the recirculation system.

FIG. 2

Identification of FA2-2 transconjugants containing pIP1017 by PFGE. Conventionally purified and BamHI-restricted total genomic DNA was analyzed by PFGE. Lanes 1 to 3, three independent clones, identified as FA2-2::pIP1017 transconjugants; lane 4, FA2-2; lane 5, OG1X; lane 6, OG1X::pIP1017. Lane 7 contained λ DNA restricted with Asp718 (30, 17, and 1.5 kb). The two arrows on the left indicate the positions of bands specific for FA2-2 genomic DNA; the arrow on the right indicates the position of a pIP1017-specific fragment.

FIG. 3

Comparative data for gene transfer efficiency in the Regensburg sewage water treatment plant. Efficiencies of conjugation for sex pheromone plasmid pIP1017, broad-host-range plasmid pIP501, and conjugative transposon Tn916 are shown. For the columns labelled pIP501 (+ pIP1017) and Tn916 (+ pIP1017) the values are values for transfer of the first genetic element from a donor strain also harboring pIP1017. Values obtained under laboratory conditions, obtained in the anoxic sludge digestor (35°C), obtained during the summer in the activated sludge digestor (20 to 25°C), and obtained during the winter in the activated sludge digestor (14 to 16°C) are indicated. Open bars indicate that no transconjugant was obtained and indicate the detection limit.

FIG. 4

Calculation of maximal transfer frequencies for pIP1017 at E. faecalis titers present in the Regensburg plant. Transconjugation rates (numbers of transconjugants per donor cell) were determined independent of the donor titer (note that the recipient cell titer was in each case 10-fold higher). Since the limit of detection of transconjugants was higher than the actual E. faecalis cell number in either the anoxic sludge digestor or the activated sludge basin, extrapolations had to be made to obtain calculated transconjugation rates for total E. faecalis titers of 10³ (activated sludge basin) and 10⁴ (anoxic sludge digestor).