Background Clostridium is a genus of Gram-positive, spore-forming, anaerobic bacteria comprising approximately 100 species. Some Clostridium spp. (C. botulinum, C. perfringens, C. tetani, and C. difficile) have been recognized to cause acute food poisoning, botulism, tetanus, and diarrheal illness in humans. Thus, rapid identification of Clostridium spp. is critical for source-tracking of contaminated food and to understand the transmission dynamics of these foodborne pathogens. Objective This study was carried out to rapidly identify Clostridium-like isolates by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and rRNA sequencing methods. Method Thirty-three Clostridium-like isolates were recovered from various baby food and surveillance samples. Species identification of these isolates was accomplished using the VITEK MS system. Sequence characterization of the 16S rRNA region was done on an ABI 3500xL Genetic Analyzer. Results The VITEK MS system identified 28 of the 33 Clostridium-like isolates with a high confidence value (99.9%); no identification was observed for the remaining five isolates. Nucleotide sequencing of the 16S rRNA region identified all 33 Clostridium-like isolates. Furthermore, while characterizing the 16S rRNA gene, 11 distinct Clostridium spp. (Clostridium aciditolerans, Clostridium aerotolerans, Clostridium argentinense, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cochlearium, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, and Clostridium subterminale) were recognized among the 33 Clostridium-like isolates. One of the Clostridium-like isolates was identified as Citrobacter amalonaticus by both diagnostic methods. The generated 16S rRNA sequences matched completely (100%) with sequences available in GenBank for Clostridium and Citrobacter species. Species identification attained using the VITEK MS for the Clostridium-like isolates was comparable to that from the 16S rRNA sequencing-based data. Conclusions The VITEK MS and 16S rRNA sequence analysis can be implemented in the species identification of Clostridium spp. isolates of public health importance. Highlights MALDI-TOF MS and 16S rRNA sequencing can be used in the species identification of Clostridium species.

中文翻译：

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MALDI-TOF 质谱和 16S rRNA 基因序列分析鉴定食源性梭菌属

背景梭状芽胞杆菌是革兰氏阳性、孢子形成、厌氧菌属，包括大约 100 种。一些梭菌属。（肉毒杆菌、产气荚膜梭菌、破伤风梭菌和艰难梭菌）已被认为会导致人类急性食物中毒、肉毒杆菌中毒、破伤风和腹泻病。因此，快速鉴定梭菌属。对于追踪受污染食品的来源和了解这些食源性病原体的传播动态至关重要。目的本研究采用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱和rRNA测序方法快速鉴定类梭菌分离株。方法 从各种婴儿食品和监测样品中回收了 33 株梭状芽胞杆菌样分离物。这些分离株的物种鉴定是使用 VITEK MS 系统完成的。16S rRNA 区域的序列表征在 ABI 3500xL 遗传分析仪上完成。结果 VITEK MS 系统以高置信度 (99.9%) 鉴定出 33 个梭状芽胞杆菌样分离株中的 28 个；其余五个分离株未观察到鉴定结果。16S rRNA 区域的核苷酸测序鉴定了所有 33 个梭状芽胞杆菌样分离物。此外，在表征 16S rRNA 基因时，有 11 种不同的梭菌属。(Clostridium aciditolerans, Clostridium aerotolerans, Clostridium argentinense, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cochlearium, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, 和Clostridium subterminale）在33个Clostridium-like分离物中被识别。两种诊断方法均将其中一种梭菌样分离物鉴定为柠檬酸杆菌。生成的 16S rRNA 序列与 GenBank 中可用的梭状芽孢杆菌和柠檬酸杆菌序列完全匹配（100%）。使用 VITEK MS 对梭状芽孢杆菌样分离物进行的物种鉴定与基于 16S rRNA 测序的数据的鉴定结果相当。结论 VITEK MS和16S rRNA序列分析可用于梭菌属的种属鉴定。具有公共卫生重要性的分离株。亮点 MALDI-TOF MS 和 16S rRNA 测序可用于梭菌属物种的物种鉴定。两种诊断方法均将其中一种梭菌样分离物鉴定为柠檬酸杆菌。生成的 16S rRNA 序列与 GenBank 中可用的梭状芽孢杆菌和柠檬酸杆菌序列完全匹配（100%）。使用 VITEK MS 对梭状芽孢杆菌样分离物进行的物种鉴定与基于 16S rRNA 测序的数据的鉴定结果相当。结论 VITEK MS和16S rRNA序列分析可用于梭菌属的种属鉴定。具有公共卫生重要性的分离株。亮点 MALDI-TOF MS 和 16S rRNA 测序可用于梭菌属物种的物种鉴定。两种诊断方法均将其中一种梭菌样分离物鉴定为柠檬酸杆菌。生成的 16S rRNA 序列与 GenBank 中可用的梭状芽孢杆菌和柠檬酸杆菌序列完全匹配（100%）。使用 VITEK MS 对梭状芽孢杆菌样分离物进行的物种鉴定与基于 16S rRNA 测序的数据的鉴定结果相当。结论 VITEK MS和16S rRNA序列分析可用于梭菌属的种属鉴定。具有公共卫生重要性的分离株。亮点 MALDI-TOF MS 和 16S rRNA 测序可用于梭菌属物种的物种鉴定。使用 VITEK MS 对梭状芽孢杆菌样分离物进行的物种鉴定与基于 16S rRNA 测序的数据的鉴定结果相当。结论 VITEK MS和16S rRNA序列分析可用于梭菌属的种属鉴定。具有公共卫生重要性的分离株。亮点 MALDI-TOF MS 和 16S rRNA 测序可用于梭菌属物种的物种鉴定。使用 VITEK MS 对梭状芽孢杆菌样分离物进行的物种鉴定与基于 16S rRNA 测序的数据的鉴定结果相当。结论 VITEK MS和16S rRNA序列分析可用于梭菌属的种属鉴定。具有公共卫生重要性的分离株。亮点 MALDI-TOF MS 和 16S rRNA 测序可用于梭菌属物种的物种鉴定。