Ampicillin

PIK3R1-CCDC178_pcDNA3.1(+)-CeGFP (GeneScript Biotech) overexpression plasmid contains optimized PIK3R1-CCDC178 fusion gene sequence and enhanced green fluorescent protein (eGFP). The insert was removed from the PIK3R1-CCDC178_pcDNA3.1(+)-CeGFP plasmid using NheI, BamHI, and XbaI restriction enzymes and ligated according to sticky-end ligation protocol (Thermo Fischer Scientific) to create control plasmid. PIK3R1-CCDC178_pcDNA3.1(+)-CeGFP and pcDNA3.1(+)-CeGFP plasmids were transformed into competent E. coli using heat shock. Bacteria were grown on agar plates (Fisher BioReagents) containing 100 µg/ml ampicillin (Fisher BioReagents) and incubated overnight at 37°C. Isolated colonies were grown in LB broth (Fisher BioReagents) overnight at 37°C and selected with 100 µg/ml ampicillin. DNA was extracted using a Nucleospin Plasmid QuickPure kit according to the manufacturer´s protocol. Plasmid purity was verified using gel electrophoresis and Sanger sequencing.

Seeds were washed with a solution of 0.05% Triton and 70% Ethanol then stratified for 4 days at 4°C. For the assay on solid plates, seeds were placed in sterile 0.1% agar solution and pipetted onto solid Murashige Skoog (MS) growth medium (Phytotechnology Lab, Overland Park, KS, USA) with 1.2% sucrose (Fisher) and 50 mg/L ampicillin (Fisher) and allowed to grow for 5 days at 22°C under a 16 h light/8 h dark (for the initial dose-response assay) or 12 h light/12 h dark (remaining experiments) photoperiod. 15 seeds were transferred to solid MS medium containing either 0.15 or 0.3 μg/mL Tm, (Sigma-Aldrich T7765) and allowed to grow for 3 days at 22°C. The treated seeds were then transferred back to solid MS medium plates and allowed to grow for 3 days before collection for chlorophyll analysis. For the assay in liquid media, seeds were placed in sterile 0.1% agar solution and 15–20 seeds were pipetted into each well of a 12-well polystyrene plate (Fisher) containing 4 mL 0.5 × MS medium with 0.6% sucrose and ampicillin and grown for 5 days at 22°C. The MS medium was removed and replaced with fresh MS media or media containing either 0.15 or 0.3 μg/mL Tm and allowed to grow for 3 days at 22°C. The medium was again replaced with fresh MS medium and plants were allowed to grow for 3 days before collection for chlorophyll analysis. The experimental design is outlined in Figure 1.

Human adenocarcinomic alveolar basal epithelial cell line (A549) and human myeloid leukemia mononuclear (THP-1) cells were purchased from the West China Medical Center of Sichuan University and routinely preserved in our laboratory. The A549 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone, United States) supplemented with 10% fetal bovine serum (Gibco, United States), 100 IU/ml ampicillin, and 100 mg/ml streptomycin (Gibco, United States) at 37°C in a 5% CO2 humidified incubator. The THP-1 cells were maintained in RPMI-1640 (Hyclone, United States) medium supplemented with 10% fetal bovine serum (Gibco, United States), 10 mmol/L HEPES (Cellgro, United States), 100 IU/ml ampicillin, and 100 mg/ml streptomycin (Gibco, United States) at 37°C in a 5% CO2 humidified incubator. The THP-1 cells were differentiated into M0 macrophages by 100 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma, United States) stimulation for 48h, followed by 24 h rest in RPMI-1640 medium without PMA. The M0 macrophages were primed with fresh culture medium with 20 ng/ml IFN-γ (Peprotech, United States) and 1 µg/ml Escherichia coli 0111:B4 lipopolysaccharide (LPS) (Sigma, United States) for M1 polarization as previously reported by Chanput et al. (2014) (link).

To test antibiotic resistance in Campylobacter spp., the broth microdilution method was used with 5 % sheep blood. For all other pathogens, antimicrobial susceptibilities were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) Guidelines, 2015 [20 ]. All isolates of Salmonella spp. were tested for their minimum inhibitory concentrations (MICs) of ampicillin, ampicillin-sulbactam, ceftriaxone, cefotaxime, nalidixic acid, ciprofloxacin, levofloxacin, co-trimoxazole, azithromycin, chloramphenicol and tetracycline (Oxoid); DEC were tested for ampicillin, ampicillin-sulbactam, cefotaxime, ciprofloxacin, levofloxacin, chloramphenicol, tetracycline, cefazolin, cefuroxime, imipenem, amikacin and gentamicin (Oxoid); Campylobacter spp. were tested for ciprofloxacin, azithromycin, tetracycline, erythromycin and doxycycline (Oxoid); and Aeromonas spp. were tested for cefotaxime, ciprofloxacin, levofloxacin, co-trimoxazole, chloramphenicol, tetracycline, cefazolin, cefuroxime, imipenem, amikacin and gentamicin (Oxoid). ATCC 25922, 35218, 700603 and 27853 were used as quality control strains. Antibiotic susceptibility was interpreted according to CLSI guidelines, 2015 [20 ].

A loop of prepared fish samples was inoculated with TCBS agar was then incubated at 37 °C for 18–24 h [95 (link)]. The isolated colonies appeared as green or blue-green colonies (sucrose negative) according to the power to ferment sucrose [96 ]. Suspected colonies were collected and transferred onto a tryptic soya agar slant enriched with 2% NaCl for further microscopic and biochemical identification. For the isolation of A. hydrophila, Aeromonas agar base medium (Rayan) supplemented with ampicillin (5 mg/L) (Oxoid) was used. A loopful from previously incubated enriched samples of fish was inoculated on an Aeromonas agar base containing ampicillin (Oxoid) and incubated at 37 °C for 18–24 h. The isolated colonies appeared green with black centers. Suspected colonies were picked and transferred onto tryptic soya agar slants for further microscopic and biochemical identification [97 (link)].