Ann Lab Med.  2020 Jul;40(4):306-311. 10.3343/alm.2020.40.4.306.

Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar

• Uwizeyimana JD
• Kim D
• Lee H
• Byun JH
• Yong D

• KMID: 2470326
• DOI: http://doi.org/10.3343/alm.2020.40.4.306

Abstract

BACKGROUND
Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar.
METHODS
MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution.
RESULTS
The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982-1.000), P<0.001, at a cut-off point of 13 mm.
CONCLUSIONS
A reduction of the agar concentration from 100% to 30% and the addition of protamine improved colistin diffusion in agar and allowed routine colistin susceptibility testing in a clinical microbiology laboratory, but should be handled with caution.

Keyword

Colistin; Disk diffusion; Colistin susceptibility testing; Muller-Hinton agar; Protamine

MeSH Terms

Acinetobacter
Agar*
Colistin*
Diffusion*
Gram-Negative Bacteria
Microbial Sensitivity Tests
Pseudomonas aeruginosa
Sensitivity and Specificity
Agar
Colistin

Figure

• 

  Fig. 1 Change in the colistin inhibition zone diameters with commercial MHA (MHA), commercial MHA with 100 µg/mL protamine (MHAP100), MHA with 30% agar (MHA30), MHA30 with 100 µg/mL protamine (MHA30P100), and MHA30 with 150 µg/mL protamine (MHA30P150). The diffusion of colistin was improved by reducing agar concentration and protamine addition. The difference in inhibition zone diameter (mm) around colistin disks between resistant and susceptible strains became apparent in MHA30P100 and MHA30P150.

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