Fleurot Isabelle, Aigle Marina, Fleurot Renaud, Darrigo Claire, Hennekinne Jacques-Antoine, Gruss Alexandra, Borezée-Durant Elise, Delacroix-Buchet Agnès
INRA, UMR 1319 Micalis, AgroParisTech, Jouy-en-Josas, France
INRA, UMR 1319 Micalis, AgroParisTech, Jouy-en-Josas, France.
Appl Environ Microbiol. 2014 Aug;80(16):5106-15. doi: 10.1128/AEM.01042-14. Epub 2014 Jun 13.
Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.
由于细菌污染食物导致的人体中毒或感染构成了一项经济挑战和一个公共卫生问题。迄今为止，缺乏关于造成这种风险的病原体在原位的分布和表达情况的信息，这主要是因为在复杂的微生物和物理化学生态系统中检测少数细菌群体信号时存在技术瓶颈。我们用一种天然食物分离株金黄色葡萄球菌模拟了一种真正的高风险奶酪的污染情况，金黄色葡萄球菌是一种产生肠毒素的病原体，可导致食物中毒。为了克服固体基质中检测限的问题，我们选择使用一种荧光报告蛋白（超级折叠绿色荧光蛋白），它能够对金黄色葡萄球菌群体和目标基因表达进行时空监测。互补技术的结合表明，在奶酪成熟过程中，金黄色葡萄球菌优先定位于奶酪表面。免疫化学和共聚焦激光扫描显微镜使我们能够在一张图像中可视化乳制品细菌和病原体群体、毒力基因表达以及产生的毒素。该程序很容易应用于其他感兴趣的基因、其他细菌以及不同类型的食物基质。
