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评估粘菌素在琼脂中的稳定性,并比较四种粘菌素 MIC 检测方法。

Evaluation of colistin stability in agar and comparison of four methods for MIC testing of colistin.

机构信息

Laboratory of Medical Microbiology, Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.

Department of Microbiology, Medical School, University of Athens, Athens, Greece.

出版信息

Eur J Clin Microbiol Infect Dis. 2018 Feb;37(2):345-353. doi: 10.1007/s10096-017-3140-3. Epub 2017 Nov 25.

Abstract

Susceptibility testing for colistin remains challenging primarily due to its inherent properties. We evaluated colistin stability in agar and reproducibility of colistin MICs obtained by agar dilution, broth macro- and micro-dilution and MIC gradient strips on 3-7 iterations of each method using clinical Klebsiella pneumoniae (susceptible-CS, and resistant-CR, n = 2 each), mcr-harboring Escherichia coli (n = 2), and reference strains E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853. MICs for reference strains were not in the given range using Etest and broth microdilution (ATCC25922, 0.125 and 4 μg/ml, respectively). MICs of CR-1 and CR-2, and of the mcr-harboring E. coli showed high concordance between agar and broth dilution varying up to one 2-fold dilution. However, remarkable variations were observed on broth dilution with CS-1 and CS-2 (MIC range 0.25-32 and 0.5-64 μg/ml, respectively); whereas for agar dilution the MIC for both CS strains was 0.5 μg/ml in all the runs. MICs obtained by MIC gradient strips were lower than those obtained by dilution methods (1-2 dilutions for CS and mcr strains, and up to five dilutions for CR strains). To confirm uniform distribution of colistin in agar, a single strain was spotted in five different regions of the same plate. All spots showed concordant growth with maximum one dilution difference. No effect on MIC was found due to storage of colistin-containing agar plates for 7 days at 4 °C. In our hands, agar dilution was superior in terms of reproducibility and robustness, compared to broth dilution methods, for colistin MIC determination.

摘要

由于其固有特性,粘菌素的药敏试验仍然具有挑战性。我们评估了琼脂中粘菌素的稳定性,以及在 3-7 次重复试验中,通过琼脂稀释法、肉汤宏稀释法和微稀释法以及 MIC 梯度条法获得的粘菌素 MIC 的重现性,使用临床分离的肺炎克雷伯菌(敏感 CS 和耐药 CR,各 2 株)、携带 mcr 的大肠杆菌(2 株)以及参考菌株大肠埃希菌 ATCC25922 和铜绿假单胞菌 ATCC27853。使用 Etest 和肉汤微量稀释法,参考菌株的 MIC 不在规定范围内(ATCC25922 分别为 0.125 和 4μg/ml)。CR-1 和 CR-2 以及携带 mcr 的大肠杆菌的 MIC 在琼脂和肉汤稀释法之间高度一致,变化范围最多为 2 倍稀释。然而,在肉汤稀释法中,CS-1 和 CS-2 之间观察到显著差异(MIC 范围分别为 0.25-32 和 0.5-64μg/ml);而对于琼脂稀释法,两种 CS 株的 MIC 在所有试验中均为 0.5μg/ml。MIC 梯度条法获得的 MIC 低于稀释法(CS 和 mcr 株为 1-2 倍稀释,CR 株最多为 5 倍稀释)。为了确认粘菌素在琼脂中的均匀分布,将单株菌点种在同一平板的五个不同区域。所有点均显示出一致的生长,最大差异为一个稀释度。将含粘菌素的琼脂平板在 4°C 下储存 7 天,对 MIC 无影响。在我们的研究中,与肉汤稀释法相比,琼脂稀释法在重复性和稳健性方面更适合用于确定粘菌素 MIC。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/13d9/5780530/f7fa2cf5e45d/10096_2017_3140_Fig1_HTML.jpg

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