Disk elution method.

The DE method was performed with 2 ml of MH broth added to a sterile culture tube, followed by addition of 1 disk of ATM (BD, BBL, Sensi-Disc) and 1 disk of CZA (BD, BBL, Sensi-Disc). The tube was incubated at room temperature for 30 min to allow the antimicrobials to elute from the disk. A 0.5 McFarland standard inoculum was prepared by suspending fresh colonies from an overnight sheep’s blood agar plate in normal saline. Then, 12 μl of this suspension was added to the tube with the eluted disks and vortexed to reach a final inoculum of around 1.5 × 10⁵ CFU per ml. The results were read visually in comparison to a clear broth control after a 16- to 20-hour incubation at 35°C (Fig. 1C). We compared single-disk and double-disk elution in 2 ml of MH broth with representative isolates tested over a 3-day period and found no difference in results. Thus, single-disk testing was used (Table S3). A strain was considered synergy positive if it was ATM and CZA resistant, as determined by mBMD, with no growth observed in the tube with the combination of ATM and CZA disks (Fig. 1C). The tubes with an ATM or a CZA disk alone were observed for informational purposes but were not needed to be set up for interpretation of the results.