Disk Diffusion Susceptibility Testing for the Rapid Detection of Fluconazole Resistance in Candida Isolates
2021; 41(6): 559-567
Ann Lab Med 2020; 40(4): 306-311
Published online July 1, 2020 https://doi.org/10.3343/alm.2020.40.4.306
Copyright © Korean Society for Laboratory Medicine.
1Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea; 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
Correspondence to: Jung-Hyun Byun, M.D.
Department of Laboratory Medicine, Gyeongsang National University Hospital, Gyeongsang National University College of Medicine, Jinju 52727, Korea
Tel: +82-55-750-8423 Fax: +82-55-762-2696. E-mail: email@example.com
Dongeun Yong, M.D., Ph.D.
Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Tel: +82-2-2228-2454 Fax: +82-2-364-1583. E-mail: firstname.lastname@example.org@yuhs.ac
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of
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),
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.
Keywords: Colistin, Disk diffusion, Colistin susceptibility testing, Muller-Hinton agar, Protamine
With limited new antibiotic classes in the drug development pipeline, the global challenge of antimicrobial resistance, particularly in the treatment of multidrug-resistant (MDR) gram-negative (GN) bacteria, remains critical. Colistin, also known as polymyxin E, has become a last-resort antibiotic for the management of MDR GN bacteria [1,2]. Colistin, a member of an old class of cationic, cyclic, polypeptide antibiotics was first introduced in Japan in 1947 from the soil bacterium
Although its use was abandoned in the 1980s because of concerns of nephrotoxicity and neurotoxicity , intravenous injection of colistin sulfate was demonstrated to be safe and could be considered for the management of severe infections from sensitive MDR GN pathogens . Therefore, beginning in the mid-1990s, the clinical use of polymyxins was revived, with a focus on colistin because of its rapid bactericidal effect, broad-spectrum activity against MDR GN pathogens, and the lack of novel antibiotics against the most prevalent MDR GN bacteria [3,5,6,7,8,9,10]. The global colistin resistance rate is less than 10%, but is increasing. Increased resistance has been reported in Mediterranean and Southeast Asian countries .
The increasing use of colistin over the past several years has necessitated rapid, accurate, and reliable
This study aimed to develop a simple disk diffusion test method for colistin susceptibility testing by modifying the commercial Mueller-Hinton medium to improve colistin diffusion in agar.
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 .
First, an optimum agar concentration was determined using four strains:
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.
In total, 60 GN clinical isolates obtained from Severance hospital, including
Quality control was performed using
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.
Colistin diffusion was optimized using MHA30, because we could not use the MHA with 20% or 10% agar (data not shown). At 30% agar concentration, the addition of protamine inhibited bacterial growth and enhanced colistin diffusion. Colistin diffusion in the agar improved with protamine supplementation and a reduction in the agar concentration Su. Inhibition zone diameters on MHA30P100 and MHA30 supplemented with 150 µg/mL of protamine (MHA30P150) were similar for all bacterial isolates tested. MHA30P100 was determined as the optimal medium because bacterial growth was hampered at higher protamine concentrations (Supplemental Data Table S1).
None of the strains grew on MHA30 with ≥300 µg/mL of protamine. However, colistin-susceptible ACB strain,
We could not discriminate between colistin-susceptible and -resistant strains using commercial MHA. The categorical agreements between MICs determined by BMD were 100% in
The sensitivity and specificity of colistin disk diffusion testing by using MHA30P100 were 94.7% and 100%, respectively (Table 3). By analyzing the AUC, we found that MHA30P100 medium allowed the best discrimination between susceptibility and resistance in both
During colistin MIC determination, three ACB strains showed discrepancies of >8 µg/mL between MICs using BMD and disk diffusion, and were retested twice using glass tubes to rule out false resistance finding due to colistin binding to polystyrene wells, which has been previously reported [22,23]. Two strains were susceptible to colistin in reference BMD methods, resulting in the same category with disk diffusion tests. However, one strain remained colistin resistant, which was retested using MHA30P100 directly from glass tubes containing 0, 0.5, 1, 2, 4, or 8 µg/mL of colistin. The bacterial isolate was sub-cultured from each tube after MIC measurement and further identified as
The main objective of our study was to optimize MHA for improved colistin diffusion. We first modified the agar concentration, and optimized the medium composition with protamine (Fig. 1). When protamine was added to MHA30, colistin diffusion further increased, resulting in larger inhibition zones and a clear distinction between resistant and susceptible strains (Fig. 1). The addition of protamine to MHA30 resulted in the same sensitivity and specificity, but facilitated the interpretation of the results because the differences in inhibition zone diameters around the colistin disks on MHA30P100 were increased (Fig. 1, Table 1, and Table 3). Although protamine has bacterial growth inhibition properties , bacterial growth was not inhibited at 100 µg/mL, while colistin diffusion increased; therefore, this concentration was used in further experiments as an optimal concentration .
Three ACB strains were resistant in BMD tests using polystyrene plates, but susceptible in disk diffusion tests using MHA30 and MHA30 P100. When BMD tests for these three strains were repeated using glass tubes, two strains were susceptible to colistin in line with the disk diffusion test results. However, one strain was resistant. To find the reason, the bacterial isolate was sub-cultured from each tube after MIC measurement. Interestingly, the isolates identified from the 0.5–1 µg/mL colistin tubes were susceptible, whereas those from the 2–8 µg/mL colistin tubes were resistant (data not shown). These findings implicated that A. nosocomialis can mutate
Colistin diffusion improved in MHA30P100 and therefore, this medium can be a useful tool for detecting colistin resistance. This simple medium was easy to prepare and allowed identifying colistin-resistant isolates of both
A limitation of MHA30P100 is that it is softer than commercial MHA. Therefore, it needs to be handled with caution to avoid scratches or crumpling. Some strains of
In summary, this study demonstrated that reducing the agar concentration to 30% of the concentration in commercial MHA dramatically improved colistin diffusion and resulted in reliable colistin susceptibility testing. This modified MHA is expected to be useful in clinical microbiology laboratories for colistin susceptibility testing.
Effect of protamine at various concentrations on bacterial growth and inhibition zone diameters (mm) around the colistin disk on MHA with 100% and 30% agar concentrationsalm-40-306-s001.pdf
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.