Identification and sub-typing of food-born bacteria from animal feed ingredients, animal feed & pet foods samples using Matrix-Assisted Laser Desorption & Ionization Time-of-Flight Mass Spectrometry.

FDA CVM VET-LRN认证（U18）资助提案 对于FOA # PA-13-244，CFDA 93，103 项目概要/摘要 本项目将使用基质辅助激光解吸电离飞行时间质量 质谱法（MALDI-TOF MS）用于鉴定人类食源性病原体，包括 沙门氏菌属、E.大肠杆菌，弯曲杆菌属，单核细胞增生李斯特菌，产气荚膜梭菌， 和金黄色葡萄球菌。第一年 该项目的重点是使用MALDI- 在以下四种情况下，同样的方法将用于其他食源性病原体 年宠物食品/动物饲料的沙门氏菌污染导致人类 沙门氏菌病。然而，动物饲料成分、动物饲料和宠物零食的污染 食物还没有被系统地研究过。常规培养是“金标准”方法 用于分离和鉴定。这种方法是劳动密集型的、昂贵的和耗时的， 在分发受污染动物之前不能及时提供信息 食物基质因此，快速可靠的鉴定和分型的替代方法 迫切需要沙门氏菌的细菌，以提高动物饲料供应的安全性和完整性， 这反过来将限制人类感染。在本申请中，我们提出开发方法， 应用MALDI-TOF MS对沙门氏菌进行快速特异性鉴定和分型 在来自动物饲料基质的初步培养板上生长。微生物鉴定，使用 该技术基于直接从细胞获得的光谱图案的比较， 使用模式识别算法对照参考光谱进行细胞提取。该技术 目前以商业平台的形式提供， 证明了其用于沙门氏菌属细菌的鉴定和分型的实用性。 我们已经成功地使用该技术鉴定粪便中的沙门氏菌， 动物组织样本然而，缺乏关于使用MALDI-TOF的知识 用于鉴定来自宠物食品和动物饲料基质的细菌分离物的MS。拟议 这项研究将通过以下三个具体目标解决这些问题： 具体目标1：从各种动物中分离的食源性细菌的鉴定和分型 食品基质使用MALDI-TOF MS使用一种新的图书馆。 具体目标2：直接从生长的菌落中鉴定食源性细菌并进行亚型分型 在来自动物饲料和食物基质的各种培养基上。 具体目标3：将这一新方法转移和培训给其他Vet-LIRN laboratories. 该项目的成功完成将扩大该技术目前的应用范围， 粪便和动物组织样品添加到动物饲料基质中，以提高 美国动物饲料供应。此外，多个实验室的合作验证， 该方法将提高Vet-LIRN实验室的能力和容量，以服务于CVM的 在应对动物食品污染事件时，需要快速周转时间。

FDA CVM VET-LRN PROGRAM (U18) GRANT PROPOSAL FOR FOA # PA-13-244, CFDA 93, 103 Project Summary/Abstract This project will use Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) for identification of human foodborne pathogens, including Salmonella, E. coli, Campylobacter species, Listeria monocytogenes, Clostridium perfringens, and Staphylococcus aureus in animal feed ingredients, pet foods and animal feed. The first year of the project will focus on identification and subtyping of Salmonella bacteria using MALDI- TOF MS. The same approach will be used for other foodborne pathogens in the following four years. Salmonella contamination of pet foods/animal feeds has resulted in outbreaks of human Salmonellosis. However, contamination of animal feed ingredients, animal feed, and pet treats and foods have not been systematically studied. Routine culture is the "gold standard" method for isolation and identification. This method is labor intensive, expensive, and time consuming, which does not provide timely information before the distribution of the contaminated animal food matrices. Therefore, alternative methods for rapid and reliable identification and subtyping of Salmonella bacteria are urgently needed to improve safety and integrity of animal feed supply, which in turn will limit human infections. In this application we propose to develop methods using MALDI-TOF MS for rapid and specific identification and typing of Salmonella bacteria grown on preliminary culture plates from animal feed matrices. Microbial identification using this technology is based on the comparison of spectral patterns obtained directly from cells or cell extracts against reference spectra using a pattern recognition algorithm. This technology is currently available in the form of commercial platforms, and a considerable amount of studies have been generated demonstrating its utility for identification and typing of Salmonella bacteria. We have successfully used this technology for identification of Salmonella bacteria in fecal and animal tissue samples. There is, however, a lack of knowledge regarding the use of MALDI-TOF MS for identification of bacterial isolates from pet food and animal feed matrices. The proposed study will address these issues by the following three specific aims: Specific aim 1: Identification and subtyping of foodborne bacterial isolates from various animal food matrices using MALDI-TOF MS using a novel library. Specific aim 2: Identification and subtyping of foodborne bacteria directly from colonies grown on various culture media from animal feed and food matrices. Specific aim 3: Transferring and training of this new methodology to other Vet-LIRN laboratories. Successful completion of this project will expand the current application of this technology from fecal and animal tissue samples to animal feed matrices to improve the safety and integrity of the US animal feed supply. In addition, the multi-laboratory collaboration validation of this methodology will increase Vet-LIRN laboratory capability and capacity in order to serve CVM's need for rapid turnaround times when responding to animal food contamination events.