Iso sensitest plates

To assess fitness cost, pairwise growth competition experiments in Davis minimal medium with 25 mg/mL glucose (DM25) were performed using isolates of lineage A sampled at 2 weeks and 2 months, respectively, the latter which had acquired the plasmid closely resembling pHUSEC41-1 (Künne et al., 2012 (link)). The experiment was performed as previously described (Enne et al., 2005 (link)), but in brief, the two isolates were grown overnight in nutrient broth, and then inoculated into DM25 at a dilution of 1:10⁴ and grown for 24 h. The cultures were then mixed together in a ratio of 1:1, and then diluted 1:100 into fresh DM25. The serial passage step was continued for 6 days, corresponding to ~60 generations of competition. After initially mixing the two cultures together, and after each 24 h period, the cultures were diluted appropriately and 100 μL were added to Iso-Sensitest plates (Oxoid, Sweden) in triplicate, with and without 50 mg/L of ampicillin. Colonies were counted after over-night incubation at 37°C, where the mean number of colonies on ampicillin plates was subtracted from the plates without ampicillin to determine the mean number of colonies lacking the pHUSEC41-1-like plasmids. Six replicates of the fitness experiment were conducted.

Antimicrobial susceptibility testing was performed for a range of clinically relevant antimicrobials with different MOAs (Table 1). MICs were determined by Etests (bioMérieux) according to the manufacturer’s instructions. Briefly, pure bacterial cultures were diluted in saline to an 0.5 MacFarland standard, 100 μl of solution was inoculated and spread onto Iso-Sensitest plates (Oxoid; CM0471), and an Etest strip was placed on top. Plates were incubated for 16 to 18 h at 37°C before the result was read.