Extended Data Fig. 1: Emergence and spread of antibiotic resistance in bacteria using P2 as a model for conjugation.

a, Antibiotic resistance in bacteria can emerge through mutation, or be acquired via horizontal gene transfer. Plasmid transfer is an important driver of the spread of antibiotic resistance. Tolerance increases the abundance of bacteria that survive antibiotic exposure, allowing for a higher probability of the emergence of mutations that lead to resistance¹¹. We hypothesize that antibiotic resistance can also spread through the formation of reservoirs of persisters that contain plasmids; here, we hypothesize that the gut mucosa of the host can serve as a reservoir for persisters. The formation of long-term reservoirs, followed by re-seeding of bacteria from this reservoir into a niche occupied by other bacteria (for example, the gut lumen occupied by the microbiota following antibiotic therapy) increases the chance that two different strains interact with each other, leading to plasmid transfer (that is, increased strain co-occurrence). The representation in the bottom right panel is an example of donor persisters boosting co-occurrence. Note that tissue-associated recipient persisters may also increase co-occurrence. b, P2 shares homology with resistance plasmids. An alignment is shown between S. Typhimurium SL1344 P2 (GenBank sequence identifier HE654725.1), S. Typhimurium plasmid R64 (GenBank sequence identifier AP005147.1), and pESBL15 of E. coli Z2115 (strain isolated from a rectal swab of a patient at the University Hospital Basel) using the Artemis comparison tool (https://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act). Red fill indicates high sequence identity (>85% sequence identity), blue fill indicates inversions and no fill indicates no sequence identity. For each plasmid, open reading frames (in each of the six translational frames) are shown by white regions (detected by the Artemis comparison tool). Antibiotic resistances (for example, streptomycin and tetracycline resistances on R64 and CTX-M-1 on pESBL15) are labelled, shown by light-blue directed rectangles (found by a Basic Local Alignment Search Tool (NCBI) search against the ResFinder antibiotic-resistance-gene database⁴¹). In P2, the locus for insertion of the chloramphenicol-resistance cassette and neutral sequence tags is shown. For each alignment, the percentage of the sequence that aligns to P2 is shown, as well as the average sequence similarity for these regions. c, Model strains for addressing evolution by conjugation in S. Typhimurium. SL1344 contains P2^cat (chloramphenicol-resistance cassette (cat) that allows the enumeration of plasmid-bearing strains by selective plating) that can be conjugated to 14028S (kanamycin-resistance cassette (aphT) used for selective plating) to form a transconjugant (Cm^R and Kan^R). Transconjugants can then transfer P2^cat to additional recipients. d, P2^cat transfer kinetics in vitro. P2^cat transfer is dependent on the density of donors and recipients. Donor and recipient strains were inoculated into LB (n = 2, 1 experiment) at a 1:1 ratio and selective plating was performed every hour. CFUs per millilitre are reported for each population (donors in blue, recipients in green and transconjugants in red). Solid lines connect medians. The dotted line indicates the detection limit by selective plating.

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