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

Affiliations
  • 1Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea. microbyun@gmail.com, deyong@yuhs.ac
  • 2Department of Global Health Security, Yonsei University Graduate School of Public Health, Seoul, Korea.
  • 3Department of Emergency Care, Ruli Hospital, Gakenye, Rwanda.
  • 4Brain Korea 21 plus Program for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
  • 5Department of Laboratory Medicine, Gyeongsang National University Hospital, Gyeongsang National University College of Medicine, Jinju, Korea.

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
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