Novel sex-pilus-specific bacteriophages reduce plasmid carriage and transfer in vitro

Antibiotic resistance genes are often encoded on conjugative plasmids, which facilitate the horizontal transfer and spreading of these genes. Bacteriophages that are targeting the sex pilus of the conjugational apparatus might therefore be useful to remove resistances from bacterial populations by (i) specifically targeting and lysing bacteria that possess a plasmid-encoded sex pilus and (ii) reducing the horizontal transfer of these plasmids.

We have identified two novel bacteriophages, ɸ4187/61 and ɸ4187/77, by a spot-assay screening of a collection of phages isolated from wastewater, that are specifically targeting plasmids belonging to Inc P, N and W. Deletion of the transfer genes from the plasmids rendered the bacteria resistant to ɸ4187/61 and ɸ4187/77, indicating that the conjugation apparatus is indeed the receptor of the phages. We show that both bacteriophages reduce the amount of plasmid-harboring bacteria and drive the selection towards plasmid-free bacterial populations in in vitro co-cultivation studies. Furthermore, we show that both bacteriophages also reduce the horizontal transfer of the plasmids in vitro. In addition to that, we have analyzed resistant bacterial isolates after phage treatment and could show that plasmid loss is one of the major phage resistance mechanisms. A second sub-population of phage-resistant bacteria still contained the plasmids. However, the vast majority of plasmid-harboring and phage-resistant bacteria had lost the capability to transfer the plasmid by conjugation, whereas a minority was still able to transfer the plasmid by conjugation, albeit at a strongly reduced frequency.

In summary, we present two novel plasmid-specific bacteriophages ɸ4187/61 and ɸ4187/77 that are specifically targeting the conjugational apparatus of plasmids. We show here that these bacteriophages can be used to drive bacterial populations towards plasmid loss and to reduce the horizontal distribution of these plasmids transfer in vitro. Future experiments using suitable animal models will show, if ɸ4187/61 and ɸ4187/77 are also suitable to remove plasmids from bacterial populations in vivo.