Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies
Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies
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DOI：
10.1128/aem.02005-09
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发表时间：
2010-01-15
影响因子：
4.4
通讯作者：
Johnson, Eric A.
中科院分区：
文献类型：
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作者：
Bradshaw, Marite;Marshall, Kristin M.;Johnson, Eric A.
肉毒梭菌产生已知的最有毒的天然毒素，由于食源性肉毒杆菌中毒的潜在威胁，它是食品行业和监管机构长期关注的问题。为了确保食品的肉毒杆菌安全，经常进行严格的食品挑战检测，以验证食品加工条件和食品配方。肉毒杆菌神经毒素的检测通过使用小鼠生物检测和/或体外检测来执行。食品行业和监管机构对在这些挑战研究中最大限度地减少甚至取代动物的使用一直很感兴趣。此外，由于严格的选择试剂规定，使用肉毒杆菌毒素菌株对各种食品进行测试需要获得这种微生物工作认证的设施和人员。为此，我们建议从蛋白水解组和非蛋白水解组中产生几组不产生肉毒杆菌毒素的菌株，这些菌株与野生型菌株的不同之处仅在于它们不能产生肉毒神经毒素。在这项初步研究中，我们描述了利用Clostron突变系统通过将第二组内含子插入A型肉毒杆菌神经毒素基因(BONT/A)来产生肉毒杆菌62A菌株的非产毒突变株。通过Western blotts和小鼠生物测定，突变克隆是无毒的，但表现出与亲本菌株相似的生理特性，包括生长特性和产孢量。将需要更多的研究来评估各种食品基质中的可比特性。获得合适的无毒肉毒杆菌菌株用于食物挑战研究，将有利于提高食品的肉毒杆菌安全性，增加工作人员的生物安全性，并可能取消使用实验动物。
Clostridium botulinum produces the most poisonous natural toxin known and is a perennial concern to the food industry and to regulatory agencies due to the potential threat of food-borne botulism. To ensure the botulinal safety of foods, rigorous food challenge testing to validate food-processing conditions and food formulations has been routinely performed. Detection of the botulinum neurotoxin is performed by using a mouse bioassay and/or in vitro assays. There has been considerable interest by the food industry and regulatory agencies in minimizing or even replacing the use of animals in these challenge studies. In addition, due to stringent select-agent regulations, the testing of various foods using toxigenic C. botulinum strains requires facilities and personnel that are certified for work with this organism. For this purpose we propose to generate sets of nontoxigenic C. botulinum strains from proteolytic and nonproteolytic groups that differ from the wild-type strains only by their inability to produce botulinum neurotoxin. In this initial study we describe the generation of a nontoxigenic mutant of C. botulinum strain 62A using the ClosTron mutagenesis system by inserting a group II intron into the botulinum neurotoxin type A gene (bont/A). The mutant clones were nontoxigenic as determined by Western blots and mouse bioassays but showed physiological characteristics, including growth properties and sporulation, that were similar to those of the parent strain in laboratory media. Additional studies will be required to evaluate comparable characteristics in various food matrices. The availability of suitable nontoxigenic C. botulinum strains for food challenge studies will be beneficial for enhancing the botulinal safety of foods as well as increasing the biosafety of workers and may eliminate the use of laboratory animals.