Microbiology Animal Food Project Testing Track 3 Accelerated Sample Preparation for Microbial Pathogen Detection in Pet Food Samples

微生物病原体大批量检测的快速样品制备 宠物食品样品 摘要 我们正在提交一份关于学科A：微生物学，第3轨道动物食品产品的提案 用狗测试将食物/沙门氏菌视为对商品对的危害。在这里我们将联合收割机 来自2个实验室的不同专业知识，可再生资源工程实验室（LORRE） 和印第安纳州化学家办公室（OICS），以收集一个建议，包括动物食品 样品收集，加速食品样品制备，将感兴趣的微生物 检测水平，FDA批准的沙门氏菌环介导等温扩增（LAMP） 动物性食品中的筛选，以及全基因组测序作为病原体确认的方法 分离株作为2015年FDA的后续行动，我们进一步优化了样品制备方法。 挑战奖，我们现在可以同时集中和回收微生物， 对4个样本感兴趣，使其数量从最初无法检测到，由于存在于 初始水平为1CFU/g或更低，达到检测水平。这种样品的加速处理是 在大约4小时的时间内完成。它由微生物短富集选择性 培养基与酶处理组合，然后进行预过滤步骤以去除大的 颗粒和微滤浓缩和回收选定的微生物进行检测 水平酶处理有助于分解可能的生物膜形成和颗粒， 可能在微滤过程中堵塞中空纤维膜中的孔。选择的酶是基于 在食物基质上。在本文提出的样品的情况下，蛋白酶可能与 将使用脂肪酶。我们已经证明，当在微生物培养基中时，酶不影响微生物的活力。 它们各自的基质的存在。虽然我们关注的是沙门氏菌作为一种微生物 有趣的是，我们开发的方法也可以应用于其他微生物。办公室 印第安纳州化学家（OISC）微生物学实验室在样本收集方面拥有丰富的专业知识 用于动物食品测试。在微生物方法方面也有多年的经验 开发、抗生素浓度检测、细菌计数和鉴定， 包括几年的沙门氏菌，单核细胞增生李斯特菌， 弯曲杆菌和大肠杆菌（STEC）在动物饲料。去年，OISC微生物学 一个实验室在宠物处理猪耳中沙门氏菌全国性爆发中进行了测试， 印第安纳州。此处拟检测的样本数量为250个配对犬样本 治疗/沙门氏菌。预期的结果是加速样品制备，提高速度， 样品检测的灵敏度，以及对潜在回收和检测困难的初步认识 培养微生物。

Accelerated Sample Preparation for Microbial Pathogen Detection in Large Number of Pet Food Samples Abstract We are submitting a proposal for Discipline A: Microbiology, track 3 for Animal Food Product testing with Dog treat food/Salmonella as a hazard to Commodity pairs. Here we combine different expertise from 2 laboratories, Laboratory of Renewable Resources Engineering (LORRE) and the Office of Indiana State Chemist (OICS), to assemble a proposal consisting in animal food sample collection, accelerated food sample preparation to bring microorganisms of interest to detection levels, FDA approved loop mediated isothermal amplification (LAMP) for Salmonella screening in animal food, and whole genome sequencing as a method for confirmation of pathogen isolates. We have further optimized our sample preparation approach, as a follow up to 2015 FDA challenge award, and we now can simultaneously concentrate and recovery microorganisms of interest in 4 samples, bringing their numbers from initially not detectable, due to be present at initial levels of 1CFU/g or lower, to detection levels. This accelerated processing of samples is complete in a time frame of about 4 hours. It consists of a microbial short enrichment in selective medium combined with an enzyme treatment, followed by a pre-filtration step to remove large particles and microfiltration to concentrate and recovery selected microorganisms to detection level. The enzyme treatment helps to break down possible biofilms formation and particles that may clog pores in the hollow fiber membrane during microfiltration. Enzymes selected are based on food matrices. In the case of the samples proposed here, proteases possible combined with lipases will be used. We have shown the enzymes do not affect microbial viability, when in the presence of their respective substrates. While we have focused on Salmonella as a microorganism of interest, our developed approach can also be applied to other microorganisms. The Office of Indiana State Chemist (OISC) microbiology laboratory has major expertise in sample collection for animal food testing. It has also many years of experience in microbiological method development, testing for antibiotic concentration, and bacteria enumeration and identification, including several years of pathogen screening for Salmonella, Listeria monocytogenes, Campylobacter and Escherichia coli (STEC) in animal feed. Last year, the OISC microbiology laboratory contributed for testing in a national wide Salmonella outbreak in pet treat pig ear in Indiana. The proposed number of samples to be tested here is 250 samples pairing dog treat/Salmonella. Expected outcomes are accelerated sample preparation, increased speed and sensitivity of sample testing, and initial insights of potential recovering and detection of difficult to culture microorganisms.