Colistin col

Minimum inhibitory concentration (MIC) defined as the lowest compound concentration (μg/ml) required to stop bacterial growth was determined by using the microbroth dilution method according to the Clinical and Laboratory Standard Institutes (CLSI) recommendations (Clinical Laboratory Standards Institute, 2016 ). An ESBL-positive strain of KP ATCC700603 was used as a reference control. Three antimicrobial agents were tested: Colistin (COL, Sigma-Aldrich, St. Louis, MO), Meropenem (MEM, Haibin Pharmaceutical Co., Shenzhen, China), and amikacin (AMK, Sui Cheng Pharmaceutical Ltd., Tianjin, China). The concentration ranges of COL, MEM and AMK used in this study were 0.0625–64, 0.5–256, and 0.03125–16,384 μg/ml, respectively. The MEM and AMK results were interpreted per CLSI criteria (Clinical Laboratory Standards Institute, 2016 ), whereas the COL results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint recommendations (EUCAST, 2016 ). The MICs were determined by measuring optical density (OD) at 570 nm using a microplate reader.

Edwardsiella tarda PPD130/91 was recognized as Edwardsiella piscicida PPD130/91 ever since 2015 (Ling et al., 2000 (link); Shao et al., 2015 (link)). The E. piscicida strains and plasmids used in this study are described in Table 1. E. piscicida strains were grown in TSB medium static. To activate T6SS and T3SS, E. piscicida and its derived strains were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) at 25°C under 5% (v/v) CO₂ atmosphere. When required, appropriate antibiotics were supplemented at the concentrations of 100 μg/ml ampicillin (Amp; Sigma), 12.5 μg/ml colistin (Col; Sigma), 34 μg/ml chloramphenicol (Cm; Amresco), 15 μg/ml tetracycline (Tet; Amresco), and 50 μg/ml kanamycin (Km; BioFroxx).

Minimum inhibitory concentrations (MICs) were determined by the agar dilution method according to recommendations and breakpoints proposed by the CLSI (CLSI, 2017 ). The antibiotics assayed were imipenem (IPM), ertapenem (ETP) (Merck Sharp & Dohme Corp., Kenilworth, NJ, USA), meropenem (MER) (Sigma-Aldrich, St Louis, MO, USA), ceftazidime (CAZ), cefotaxime (CTX), ciprofloxacin (CIP), gentamicin (GEN), and amikacin (AMK) (Merck Sharp & Dohme Corp., Kenilworth, NJ, USA). MICs of colistin (COL) (Sigma-Aldrich) and tigecycline (TIG) (Pfizer, Philadelphia, PA, USA) were determined by the broth microdilution method, using the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2017 ) guidelines and break-points since breakpoints for these two antibiotics are not supplied by the CLSI.