Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles

The mechanism underlying bacterial conjugation through protozoa was investigated. Kanamycin-resistant Escherichia coli SM10λ⁺ carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. coli clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Page’s amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10^?6, but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10^?8. Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.