Copper, gentamicin, and oxytetracycline susceptibility testing

Susceptibility of 13 isolates to antibacterial products was evaluated by the broth dilution method (Wiegand, Hilpert & Hancock, 2008).

Tested isolates were selected based on the highest disease severity evidenced in the pathogenicity test, as well as five strains with medium, and other four showing low severity. The evaluated products were Glucob Plus^® (8% copper gluconate + 1% citric acid (CuG)), Coboxy^® (45% copper oxychloride + 37.7% oxytetracycline hydrochloride (Cu + Ox)), Final Bacter (2% gentamicin + 6% oxytetracycline hydrochloride (Gen + Ox)), gentamicin sulphate (GenS), and copper sulphate pentahydrate (CuS). For minimum inhibitory concentration (MIC) analysis, antimicrobial dilutions were prepared at 4, 8, 16, 32, 64, 128, 256, 512, and 640 µg/mL in tryptone soy broth (TSB). Microbial resistance was defined by Xanthomonas growth on copper compounds at 200 µg/mL (Zhang et al., 2013; Roach et al., 2020), on Ox at 25 µg/mL (Farfán, Benítez & Carvajal, 2014), and on gentamicin at 25 µg/mL (Rojas, Peña & Peña-Vera, 2019). In addition, pH values from all the antimicrobial tested and their dilutions were determined.

Bacterial suspensions were adjusted to 0.5 McFarland standard (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) by turbidimetry, which is equivalent to 1 × 10⁸ CFU/mL in TSB. Bacterial suspension was mixed and diluted to 1:100. Next, 50 μL of bacterial suspension plus 50 μL of antimicrobial solution were placed in a microplate, using 100 µL of TSB as a negative control, and 50 µL TSB + 50 µL of each antimicrobial as blank and TSB + bacterial suspension as positive control. Final inoculum contained 5 × 10⁵ CFU/mL. Microplates were then sealed with Parafilm and incubated at 28 °C for 48 h under constant shaking at 225 rpm. After incubation, ODs were read in a Varioskan Flash microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 600 nm for MICs determination. Bacterial growth was confirmed by seeding 10 µL of each treatment plus 990 µL of TSB in NA and incubating at 28 °C for 48 h. Tests were performed in triplicate.