Type of conjugative pili governs transfer efficiency in liquid and affects interpretation of transfer assays.

Plasmid conjugation is central to plasmid maintenance and spread among bacteria. Conjugation assays in liquid or on solid media are commonly used to quantify plasmid conjugation rates. Plasmids with short, rigid conjugative pili are thought to conjugate more efficiently on surfaces, whereas plasmids encoding long, flexible pili can conjugate efficiently in liquid medium. However, this pattern has not been tested systematically. Here, we perform standardised conjugation assays on a collection of 13 conjugative plasmids belonging to families that play a key role in AMR transmission and encode different conjugative pili types. We confirm that only the plasmids encoding long flexible pili conjugate efficiently in liquid. Furthermore, most transconjugants that arise from liquid assays involving plasmids with short, rigid pili can be attributed to transfer happening after the assay itself, on the surface of selective plates. This effect is amplified when using auxotrophic rather than antibiotic resistance markers, and impacts measures of transfer and defence efficiency. Finally, most of the tested plasmids with short pili had very high conjugation rates on surfaces, suggesting their transfer is mostly limited by physical constraints.