Abstract: This study aimed to establish a rapid method for identifying three common foodborne pathogenic Gram-negative bacteria (Vibrio parahaemolyticus, Enterobacter cloacae, and Acinetobacter baumannii). We explored the effects of different pretreatment methods, media, and culture durations on the results of bacterial identification using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We optimized the MALDI-TOF/TOF MS (tandem TOF MS) method with respect to culture medium and time. Formic acid-acetonitrile extraction was found to be the most suitable pretreatment method. The single-stage TOF MS spectra showed significant differences in identification scores, the number of characteristic peaks, and morphology among strains grown on different media. The number of major ion peaks decreased with culture time, leading to reduced matching accuracy. The tandem TOF MS spectra showed no significant changes in the characteristic peaks of strains at different culture times, indicating that the tandem TOF MS method was less affected by the external environment, exhibiting higher specificity. The single-stage TOF MS method had high accuracy for identifying strains cultured in specific environments for short durations. However, as the culture environment changed and the culture time increased, the results of single-stage TOF MS became unstable, while those of tandem TOF MS changed little. When they were applied to identify Gram-negative bacteria, compared with single-stage TOF MS, tandem TOF MS gave more stable results with fewer interfering peaks, thereby serving as an effective supplement to the existing microbial identification methods.

Key words: Gram-negative bacteria; matrix-assisted laser desorption ionization time-of-flight mass spectrometry; rapid identification; pathogens

摘要： 旨在建立一种快速鉴定3 种常见食源性致病革兰氏阴性菌（副溶血性弧菌、阴沟肠杆菌、鲍曼不动杆菌）的方法。采用基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption ionization time-of-flight mass spectrometry，MALDI-TOF MS）技术，探索不同预处理方法、不同培养基、不同培养时间对鉴定结果的影响。通过MALDI-TOF/TOF MS（二级TOF MS）技术优化检测方法，进一步研究不同培养基、不同培养时间的菌株对二级TOF MS鉴定结果的影响。结果表明，最适宜前处理方法为甲酸-乙腈提取法；一级TOF MS图显示，采用不同培养基培养的菌株鉴定分值和特征峰数量、形态均有明显差异；随培养时间延长，主要离子峰数量减少，导致匹配度降低。二级TOF MS图显示，实验菌株在不同培养时间主要特征峰无明显变化，二级TOF MS鉴定结果受外界环境影响较小，鉴定结果特异性更高。一级TOF MS法对特定培养环境和较短培养时间菌株鉴定有较高准确性，随着培养环境改变、培养时间延长，鉴定结果出现不稳定现象，而二级TOF MS法鉴定结果变化较小。相较于一级TOF MS鉴定，二级TOF MS技术对革兰氏阴性菌株鉴定结果稳定，杂峰较少，可作为微生物鉴定方法的有效补充。

关键词: 革兰氏阴性菌；基质辅助激光解吸电离飞行时间质谱；快速鉴定；致病菌