We screened the pathogen library for Carb, Cm, and Kan resistance to establish selection markers for conjugation quantification. The BSIB had previously collected disk diffusion data on β-lactam antibiotics other than Carb, which we used to infer Carb susceptibility (CarbS) for 51 isolates. Isolates without prior data or with ambiguous resistance were inoculated in 1 ml of LB media in 96–deep well plates, covered in a gas-permeable membrane, and grown overnight. Once grown, cultures were diluted 1000× into secondary 96-well plates with and without added antibiotic. Concentrations for antibiotics were as follows: Carb (100 μg/ml), Cm (50 μg/ml), and Kan (50 μg/ml). Secondary plates were then grown for approximately 16 hours. At this point, the OD600 of each culture was measured in a plate reader. Antibiotic culture OD was normalized to corresponding no-antibiotic cultures. Resistance to each antibiotic was defined as ≥1% maximal growth.

The MIC, which we defined as the first antibiotic concentration to yield 10% or less of the no-antibiotic control’s maximum OD600, and IC50 were also determined for plasmid donors tested for antibiotic effects on conjugation (fig. S3A and table S3). Plasmid donors were grown for 16 hours, diluted 100-fold into M9CA with antibiotic concentrations ranging from 0 to 64 μg/ml, and then grown for 24 hours before taking endpoint OD600 measurements.

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