Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
Appl Environ Microbiol. 2010 Jan;76(2):387-93. doi: 10.1128/AEM.02005-09. Epub 2009 Nov 20.
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.
肉毒梭菌产生已知最毒的天然毒素，由于食源性肉毒中毒的潜在威胁，它一直是食品工业和监管机构关注的焦点。为了确保食品的肉毒安全性，已经例行进行了严格的食品挑战测试，以验证食品加工条件和食品配方。通过使用小鼠生物测定和/或体外测定来检测肉毒神经毒素。食品工业和监管机构对尽量减少甚至取代这些挑战研究中动物的使用非常感兴趣。此外，由于严格的选择剂法规，使用产毒肉毒梭菌菌株测试各种食品需要经过认证的设施和人员才能进行该生物体的操作。为此，我们拟从蛋白水解和非蛋白水解组中生成一组非产毒肉毒梭菌菌株，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，我们描述了使用 ClosTron 诱变系统通过将 II 类内含子插入肉毒神经毒素 A 型基因 (bont/A) 来生成 62A 型肉毒梭菌的非产毒突变体。通过 Western blot 和小鼠生物测定确定突变体克隆是非产毒的，但显示出生理特性，包括生长特性和孢子形成，与亲本菌株在实验室培养基中的特性相似。需要进行额外的研究来评估各种食品基质中的可比特性。适合食品挑战研究的合适非产毒肉毒梭菌菌株的可用性将有助于提高食品的肉毒安全性，提高工人的生物安全性，并可能消除对实验室动物的使用。
