Antibiotic-Resistance Assays

Xanthomonas albilineans strains XA23 and LS126 were each cultured for 48 h at 30°C, and their optical densities at wavelength λ = 600 nm (OD₆₀₀) were estimated via spectrophotometry. Each culture was then diluted in MW medium, to obtain OD₆₀₀ = 0.25. Then, a 10-μL sample of each diluted culture was spotted on a SPA plate, allowed to dry completely and then incubated for 5 days at 30°C. SPA plates were overlaid with 4 mL of LB top agar (0.75%) with 1 mL of a test E. coli strain at OD₆₀₀ = 0.25, and incubated overnight at 37°C. The next day, plates were imaged. Images were quantified using ImageJ, by thresholding until either the X. albilineans spots or E. coli clearings were outlined and the area within the outline was quantified. The ratio of the area of the zone of clearing to the area of the X. albilineans spot was used to define albicidin sensitivity: a ratio of greater than 1.0 indicates albicidin sensitivity, while a ratio of 1.0 indicates albicidin resistance.

Minimum inhibitory concentrations (MICs) of ampicillin and ciprofloxacin were determined using the twofold dilution method (MacLowry et al., 1970).