Jung J S, Eberl T, Sparbier K, Lange C, Kostrzewa M, Schubert S, Wieser A
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Marchioninistr. 17, 81377, Munich, Germany,
Eur J Clin Microbiol Infect Dis. 2014 Jun;33(6):949-55. doi: 10.1007/s10096-013-2031-5. Epub 2013 Dec 14.
With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.
随着细菌耐药性的出现及其复杂性的增加，快速且可靠的药敏试验已成为一个热门问题。因此，有助于预测经验性抗生素治疗效果的新技术备受关注。尽管使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）快速检测抗生素耐药性是一个有吸引力的选择，但目前用于MALDI-TOF MS药敏试验的方法仅限于非常有限的条件。在此，我们描述了一种技术，该技术可能实现与表型方法相当程度的快速药敏试验。该试验基于细胞培养中氨基酸稳定同位素标记（SILAC）类似的方法。此技术用于观察在抗生素存在下细菌的生长情况。选择铜绿假单胞菌作为模式生物，将菌株分别在正常培养基、添加（13）C6 - （15）N2标记赖氨酸的培养基以及添加标记赖氨酸和抗生素的培养基中培养。通过MALDI-TOF MS检测由于标记氨基酸掺入而发生的峰位移。测试了三种作用机制不同的抗生素，美罗培南、妥布霉素和环丙沙星。创建了一种半自动算法以实现快速且无偏的数据评估。通过所提出的试验，对于所有三种抗生素都能够明确区分耐药和敏感菌株。SILAC技术在抗生素耐药性检测中的应用可能有助于加快药敏试验并提高其可靠性。
