Antibiotic discs

The antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method on Mueller Hinton Agar (Becton, Dickinson and Company, MD, USA) based on the Clinical Laboratory Standard Institute (CLSI) guidelines [18 ]. The antibiotic discs (Becton, Dickinson and Company, MD, USA) used included ampicillin (10 μg), sulfamethoxazole/trimethoprim (1.25/23.75 μg), streptomycin (300 μg), ciprofloxacin (5 μg), tetracycline (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), chloramphenicol (30 μg), ceftazidime (30 μg), norfloxacin (10 μg), and cefotaxime (30 μg). The phenotypic confirmation of ESBL isolates was done by the combination of disc approximation method using either ceftazidime (30 μg) or cefotaxime (30 μg) alone followed by overnight incubation at 37°C for 18–24 hrs. Interpretation of susceptibility patterns on other antimicrobial discs was done using guidelines laid down in the CLSI, which provides break points corresponding to zone of inhibition diameter. An increase in antibiotic zone diameter (5–12 mm) for either ceftazidime or cefotaxime indicated ESBL production [10 (link), 18 ]. Quality control standard laboratory procedures were strictly adhered to, to avoid contamination. Escherichia coli ATCC 25922 was used as a quality control organism.

The experimental procedures strictly followed the twofold dilution method recommended by the CLSI Clinical Laboratory Standardization Association to measure the MIC value of CR-KP. The final concentration gradient of each drug in the experiment was 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg/L. The inoculum machine took 1 mL of the prepared inoculum and added it to each test tube to prepare the final bacterial solution concentration of 10⁴ CFU/mL. The inoculated dilution tube was stoppered and incubated overnight in an ordinary air incubator at 35°C. The VITEK automatic analyzer was used for drug susceptibility detection. The supporting drug-sensitive cards include amoxicillin, aztreonam, compound sulfamethoxazole, cefoperazone sulbactam, ampicillin, cefepime, cefuroxime, imipenem, piperacillin, levofloxacin, amikacin, cefazolin, cefotaxime, ceftazidime, ceftriaxone, gentamicin, tetracycline, meropenem, ciprofloxacin, tobramycin, and minocycline. Antibiotic discs were purchased from Becton Dickinson, USA.

Four antimicrobial agents: cefepime (Bristol-Myers Squibb), ceftazidime (Polpharma), cefotaxime (Sigma), ceftriaxone (the Institute of Biotechnology and Antibiotics, Warsaw) and ofloxacin (Sigma), as well as EPI Phe-Arg-β-naphthylamide (PAβN; Sigma), were used in the study to determine the MIC values of antimicrobial agents ± EPI. Detection of ESβL production by Gram-negative rods was determined using antibiotic discs from Becton Dickinson. For the selection of transformants and transconjugants during construction of the P. aeruginosa PAO1161 ΔampC mutant, ampicillin (Sigma), carbenicillin (Sigma), rifampicin (Sigma), isopropyl-β-D-thiogalactopyranoside (Sigma) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Sigma) were used.