3.4. Determination of Minimum Inhibitory Concentration (MIC)

The MIC of each antibiotic was determined using the agar dilution method according to the CLSI guidelines [23,24]. According to the recommendations of the CLSI, the 9 kinds of antibiotics for the Gram-negative bacteria were tested as follows: ampicillin (ABPC, Wako Pure Chemical Industries, Osaka, Japan), cefazolin (CEZ, Wako Pure Chemical Industries), cefotaxime (CTX, Wako Pure Chemical Industries), imipenem (IPM, Wako Pure Chemical Industries), gentamicin (GM, Wako Pure Chemical Industries), tobramycin (TOB, Wako Pure Chemical Industries), ciprofloxacin (CPFX, Wako Pure Chemical Industries), chloramphenicol (CP, Sigma-Aldrich, St. Louis, MO, USA), tetracycline (TC, Wako Pure Chemical Industries). All antibiotics were dissolved and diluted according to CLSI guidelines and antimicrobial solutions were used on the day of preparation. The plates contained two-fold dilutions of antibiotics with ten-grade concentration ranging as follows: 0.25 to 128 μg/mL for ABPC and CP; 0.0625 to 32 μg/mL for CEZ; 0.03 to 16 μg/mL for CTX, IPM and CPFX; 0.125 to 64 μg/mL for GM, TOB and TC. The identified E. coli strains were cultured for 18 h in Mueller Hinton broth (MH, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Inocula were then applied to the surface of MH agar (1.7% agar) plates containing various concentrations of each antibiotic using Micro Planter (Sakuma Co., Tokyo, Japan). The plates were incubated at 37 °C for 18 h, and MICs were determined. MIC breakpoints for intermediate and resistant samples were based on CLSI criteria [24]. A reference strain of E. coli ATCC25922 was used as a quality control.