Antimicrobial susceptibility – broth microdilution

The antimicrobial susceptibility of P. aeruginosa NCIMB 10548 to ceftazidime was tested based on the broth microdilution method described by BS EN ISO 20776-1:2006 [45]. Ceftazidime was prepared at concentrations ranging from 256 mg L^− 1 to 0.25 mg L^− 1. Fifty microliters of each antibiotic concentration and a control of 0 mg L^− 1 of ceftazidime were dispensed in triplicate into wells of a 96 well multi-well plate. Overnight plate cultures were used to prepare a standardized suspension (1 × 10⁶ cfu mL^− 1) of P. aeruginosa NCIMB 10548 in 10 mL Muller-Hinton Broth (MHB) (Oxoid Ltd. Basingstoke, UK). Fifty microliters of the inoculum suspension were added to each of the wells of the multi-well plate containing 50 μL of MHB or MHB with ceftazidime. The resulting final inoculum was approximately 5 × 10⁵ cfu mL^− 1 and final ceftazidime concentrations ranged from 128 mg L^− 1 to 0.125 mg L^− 1, plus antibiotic free controls. Additionally, three wells were prepared containing 100 μL of MHB only as un-inoculated negative controls. The multi-well plate was then incubated at 37 °C for 18 h. The inoculum suspension was serially diluted and 100 μL spread plated on to TSA and incubated at 37 °C overnight to confirm appropriate inoculum preparation. Following incubation each well of the plate was visually inspected to identify turbidity to indicate growth of the test organism by comparison to the controls. Agar plates were counted after 18–24 h to confirm inoculum density was within the required range.