😂 Thanks Ross, too kind!

[14/14] pRUM-like plasmids continue to diversify through the actions of IS1216 and other small MGEs

These adaptable MDR platforms contribute to the success of CC17 E. faecium, and have the potential to enter other clones, species or genera through cointegrate formation + HGT

Thanks for reading!

[13/14] We propose a model for the IS1216-mediated evolution of the pRUM-like lineage from pCANE via an intermediate we call pMOLASSES

This seems to have involved an evolutionary trade-off, swapping transfer ability for the plasticity of the IS1216 accessory region 🤔 more on this in the paper!

[12/14] The 1990s plasmid matched the pRUM backbone almost perfectly, but it was larger and didn’t contain IS1216… instead of an accessory region, it had 44 kb that looked like backbone and contained putative conjugation determinants 🚀

This looked like the ancestor of pRUM, so we called it pCANE

[11/14] At this point the story strays into a bit of plasmid archaeology…

For her PhD, @freyaallen.bsky.social was working on a ST17 vancomycin-sensitive E. faecium from the 1990s, and she wondered, does it have a pRUM-like plasmid?

It does, but what we found surprised us! 😮

[10/14] Since 2019 I'd wondered, what did the pRUM-like lineage emerge from?

The accessory region wasn’t flanked by a target site duplication, which suggested some backbone had been lost in old IS1216-mediated deletions… but how much? What was lost, and what would an ancestral plasmid look like? 🤔

[9/14] Importantly, we saw lots of cointegrate formation, generating structures comprised of parts from usually distinct plasmids

IS26 family elements are great at this!

Several small plasmids have been rolled into pRUM-like structures, including pCOLA and pDRY, named after popular rum mixers 🍹

[8/14] Examining plasmid structures revealed enormous variation!

Most was in the accessory region, which ranged in size from 809 bp (a lone IS1216) to 285 kb, and included all sorts of acquired genes

Multiple IS1216-mediated deletions have also removed adjacent parts of the backbone

[7/14] We screened GenBank and found 150 complete pRUM-like plasmid sequences

Their mostly E. faecium hosts were isolated from various sources around the world, and represented an array of STs… though all but one were E. faecium CC17

[6/14] Over the years I spoke a lot about wanting to finish this, but the reality of side projects is it’s just hard to find the time…

This one revived in 2023 when @freyaallen.bsky.social, always interested in evolution, wanted to learn about these MGEs, giving the project the kickstart it needed!

[5/14] The pHHEf1/pHHEf2 variation had all the hallmarks of IS1216 activity, but was this the extent of it? Or was there more variation in this accessory region?

We’d need to examine more pRUM-like plasmid structures, which I started in 2019, but this project soon fell behind a lot of others…

[4/14] Why is IS1216 important? It’s part of the IS26 family!

IS26 is a major driver of MDR in Gram-negative pathogens, and I’ve worked on it or structures impacted by it across various projects since 2012...

Check out Harmer and Hall’s review to learn more: journals.asm.org/doi/10.1128/...

[3/14] Two plasmids caught my eye: pHHEf1 and pHHEf2

Their pRUM-like backbones were identical, with ARG-bearing accessory regions in exactly the same position, but the content of those regions was totally different!

Strikingly, both were bounded by copies of the insertion sequence IS1216…

[2/14] @roscobacterium.bsky.social and @wvschaik.bsky.social introduced me (a Gram-negative person) to the world of enterococci when I joined UoB in 2019…

They asked if I was interested in looking at plasmids in local clinical isolate genomes, and of course I was keen to explore Gram-positive MGEs

Thanks David!