Rapid antibiotic susceptibility testing via electrochemical measurement of ferricyanide reduction by Escherichia coli and Clostridium sporogenes
Rapid antibiotic susceptibility testing via electrochemical measurement of ferricyanide reduction by Escherichia coli and Clostridium sporogenes
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DOI：
10.1021/ac0003596
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发表时间：
2000-10-15
影响因子：
7.4
通讯作者：
Mikkelsen, SR
中科院分区：
文献类型：
--
作者：
Ertl, P;Robello, E;Mikkelsen, SR
呼吸链活性的电化学测量可以快速可靠地筛选微生物的抗生素敏感性。对有氧培养的大肠杆菌与非天然氧化剂六氰铁酸钾（III）（铁氰化物）结合的悬浮液进行时温法和时温法测定，如果大肠杆菌与有效的抗生素化合物短暂孵育，则其还原产物亚铁氰化物再氧化的信号要小得多。在Pt工作电极上，在+0.50 V vs Ag/AgCl条件下，用抗生素孵育20分钟，在含有50 mM铁氰酸盐和10 mM琥珀酸盐的实验缓冲液中孵育2分钟，获得的计时测量结果与传统的圆盘扩散敏感性试验进行比较，后者需要在琼脂平板上孵育过夜；结果表明，在所有情况下，亚铁氰化物的积累明显较低，其中生长抑制观察到在磁盘扩散试验。一系列抗生素化合物（13）被检查具有不同的作用机制。青霉素G和氯霉素的IC50值在孵育10 h后比标准浊度法的IC50值高100倍；在实验混合物中加入5 μ M 2,6-二氯苯酚（一种疏水电子转移介质），可以对革兰氏阳性专性厌氧菌芽孢梭菌进行药敏试验。这种快速的新检测方法将促进；临床药敏试验，一旦获得临床分离株，就可以进行适当的治疗。
Electrochemical measurement of respiratory chain activity allows rapid and reliable screening for antibiotic susceptibility in microorganisms. Chronoamperometry and chronocoulometry of suspensions of aerobically cultivated E. coli combined with the non-native oxidant potassium hexacyanoferrate(III) (ferricyanide) yield signals for re oxidation of the reduction product ferrocyanide that are much smaller if the E. coli has been incubated briefly with an effective antibiotic compound. Chronocoulometric results, obtained following 20-min incubation with antibiotic and 2-min measurement in assay buffer containing 50 mM ferricyanide and 10 mM succinate, at +0.50 V vs Ag/AgCl at a Pt working electrode, were compared with traditional disk diffusion susceptibility testing, which requires overnight incubation on agar plates; the results show significantly lower accumulation of ferrocyanide in all cases in which growth inhibition was observed in the disk diffusion assay. A range of antibiotic compounds (13) were examined that possess different mechanisms of action. Quantitative determination of IC50 values for penicillin G and chloramphenicol yielded values that were 100-fold higher than those obtained by standard turbidity methods after 10-h incubation; this is likely a result of the very brief (10 min) exposure time to the antibiotics, Addition of 5 mu M 2,6-dichlorophenolindophenol, a hydrophobic electron-transfer mediator, to the assay mixture allowed susceptibility testing of a Gram-positive obligate anaerobe, Clostridium sporogenes. This rapid new assay will facilitate;clinical susceptibility testing, allowing appropriate treatment virtually as soon as a clinical isolate can be obtained.