Carbenicillin

The antibiotic susceptibility of Acinetobacter baumannii isolates are based on the results of disc diffusion and minimum inhibitory concentration (MIC). The disk diffusion method is according to CLSI guidelines [16 (link)]. Eleven different antibiotics were used to assess the susceptibility test including imipenem (10 µg), cefepime, (30 µg), ceftazidime (30 µg), amikacin (30 µg), gentamicin (10 µg), tetracycline (30 µg), ticarcillin (75 µg), piperacillin (100 mg), sulfamethoxazole/trimethoprim (25 µg), carbenicillin (100 µg) and streptomycin (10 µg) (Sigma-Aldrich, St. Louis, MI, USA).
Broth dilution method was used to determine the minimum inhibitory concentration according to CLSI guidelines [16 (link)]. The antibiotics imipenem, cefepime, ceftazidime, amikacin, gentamicin, tetracycline, ticarcillin, piperacillin, sulfamethoxazole/trimethoprim, carbenicillin and streptomycin (Sigma-Aldrich) were used for MIC determination. Multidrug resistance was defined in this analysis as resistance following five drug classes: Extended-spectrum cephalosporins (ceftazidime and cefepime), beta lactamase inhibitor penicillin (ticarcillin, piperacillin and carbenicillin), aminoglycosides (amikacin, gentamicin and streptomycin), Folate pathway inhibitors (sulfamethoxazole/trimethoprim) and carbapenems (imipenem).

Glycerol stocks of recombinant A. tumefaciens were grown in 10 ml Luria broth (LB) and scaled up to 1 litre in LB base medium as previously described (Margolin et al., 2019). A. tumefaciens strains transformed with pEAQ‐HT expression plasmids were selected for using 50 µg/mL kanamycin (Sigma‐Aldrich St Louis, Missouri) and 50 µg/mL carbenicillin (Sigma‐Aldrich) whereas A. tumefaciens GV3101::pMP90RK (pTRAkc‐ERH: CHIKV E2∆TM) was selected for using 50 µg/mL rifampicin, 50 µg/mL carbenicillin and 30 µg/mL kanamycin (Sigma‐Aldrich). Growth media was supplemented with 20 µm acetosyringone in the final culture step. rifampicin was omitted from the final culture step for A. tumefaciens GV3101::pMP90RK (pTRAkc‐ERH: CHIKV E2∆TM). The bacterial inoculum was adjusted to a final OD₆₀₀ of 0.5 for each construct using resuspension medium (10 mm MgCl₂, 10 mm MES [pH5.6], 200 µm acetosyringone), except for RVFV ptGn which was previously reported to accumulate optimally at OD₆₀₀ = 0.25. Whole N. benthamiana plants were vacuum infiltrated with the bacterial suspension as reported previously (Margolin et al., 2019).

Chemical synthesis of bacteriocins BHT-B, K411, Ent7, EntL50, WelM, and SalC with a purity of >95% was performed by PepMic (People’s Republic of China). Nisin (≥900 IU/mg) was purchased from Glentham Life Sciences Ltd. (UK). Stock solutions (1 mg/mL) were prepared by dissolving lyophilized bacteriocins in 0.1% trifluoroacetic acid (TFA; Sigma, Germany). Nisin solution was additionally filtered through a 0.22-μm filter (Millipore, Germany). Gramicidin (GRA; Sigma), ramoplanin (RAM; Sigma), carbenicillin (Millipore), and chlortetracycline (Millipore) were purchased in powder form. Their stock solutions were prepared in ethanol (GRA, 1 mg/mL) or in sterile water (RAM and chlortetracycline at 1 mg/mL, carbenicillin at 100 mg/mL). Other antibiotics used in this study were purchased in the form of antibiotic-impregnated strips (bioMérieux, France; except for bacitracin [BAC], which was from Liofilchem, Italy).