Disk diffusion test
The colistin disk diffusion test was performed using a 10 mg colistin disk on Mueller-Hinton agar (MHA) plates that were incubated at 35℃ for 16–18 hours in 5% CO2. Disk diffusion test results were interpreted based on the diameter of inhibition zone and compared with MICs determined by BMD according to the 2018 CLSI guidelines .
Optimization of the agar concentration
First, an optimum agar concentration was determined using four strains: P. aeruginosa ATCC 27853, a colistin-susceptible Acinetobacter calcoaceticus-baumannii complex (ACB) strain, a colistin-resistant ACB strain, and mcr-1-harboring Klebsiella aerogenes. Assays were run in triplicate. Species were identified using a Microflex LT Biotyper (Bruker Daltonics, Leipzig, Germany). MHA was modified by reducing the agar granule concentration from 100% (17 g/L) to 30% (5.1 g/L) of the concentration in commercial MHA with 10% intervals (Becton, Dickinson, & Company, Sparks, MD, USA). The optimum concentration was determined based on the least agar concentration that was manageable in the laboratory. Lower the agar concentration, more fragile is the AST determination. The final agar concentration was reduced to 30%.
Optimization of the protamine concentration
We added protamine (Sigma-Aldrich, St Louis, MO, USA) to the modified MHA at various concentrations (1,000 µg/mL, 700 µg/mL, 400 µg/mL, 300 µg/mL, 200 µg/mL, 150 µg/mL, 100 µg/mL, and 50 µg/mL) to determine an optimal concentration that would promote colistin diffusion in agar, but not inhibit bacterial growth. Protamine was measured, mixed with distilled water until completely dissolved, and then added to the MHA before autoclaving.
Colistin MIC and inhibition zone diameter around colistin disks on modified MHA
In total, 60 GN clinical isolates obtained from Severance hospital, including P. aeruginosa (N=27) and ACB (N=33), were tested (Table 1). The study was approved by the Institutional Review Board of Yonsei University Health system, Seoul, Korea. (1-2017-0079). The clinical strains were collected from sputum and urine in 2017 and stored at −70℃.
Quality control was performed using E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and mcr-1-harboring K. aerogenes (a clinical isolate). Colistin MICs were determined by BMD for all 60 strains using colistin sulfate salt (Sigma-Aldrich, St. Louis, MO, USA) and polystyrene 96-well microplates (Corning, NY, USA). As shown in Table 1, disk diffusion AST results were interpreted based on the breakpoints, which showed good agreement with the MIC determined by BMD according to the 2018 CLSI guidelines . Each strain was tested using predetermined media, i.e., commercial MHA (100% agar concentration), MHA with 30% agar (MHA30), and MHA30 with 100 µg/mL protamine (MHA30P100), which were selected based on the optimization in phase I. The colistin disk diffusion test was performed using a 10 mg colistin disk on MHA plates that were incubated at 35℃ for 16–18 hours in 5% CO2.
Descriptive and statistical analyses were performed using SPSS 21 (Armonk, NY, USA) and MedCalc Statistical Software 18.10 (MedCalc Software, Ostend, Belgium; http://www.medcalc.org). Colistin MICs, as reference test, and disk diffusion results were compared to calculate sensitivity, specificity, and Kappa value of this simple disk diffusion test. Area under the curve (AUC) for inhibition zone diameters cutoff was determined from the receiver operating characteristic curve. P<0.05 was considered statistically significant.