Bäuerl Chrstine, Umu Özgun C O, Hernandez Pablo E, Diep Dzung B, Pérez-Martínez Gaspar
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC).
Department of Food Safety and Infection Biology, Norwegian University of Life Sciences (NMBU).
J Vis Exp. 2017 Jul 25(125):56053. doi: 10.3791/56053.
Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.
随着我们对肠道微生物群组成及其与健康关系的认识不断深入，出现了一些非常有趣的问题，特别是与维持种群平衡的因素有关的问题。然而，评估这些因素的可用方法有限。细菌素是许多细菌产生的抗菌肽，可能在获取食物和/或建立生态位方面赋予竞争优势。许多益生菌乳酸菌菌株具有通过阻止病原体生长来促进人类和动物健康的巨大潜力。它们还可用于免疫调节，因为它们能产生细菌素。然而，与人类和动物复杂的肠道环境相比，细菌素的拮抗活性通常是在明确但过于简化的条件下通过实验室生物测定来确定的，在这种肠道环境中，细菌面临来自宿主和数百种共享同一生态位的微生物物种的多因素影响。这项工作描述了一种完整而有效的程序，用于评估在小鼠系统中具有不同靶标特异性的多种细菌素的效果。使用组成性16S rDNA测序监测细菌素治疗期间微生物群组成的变化。我们的方法使用细菌素产生菌及其同基因非细菌素产生突变体，后者能够区分微生物群中与细菌素相关的修饰和与非细菌素相关的修饰。粪便DNA提取和16S rDNA测序方法是一致的，并且与生物信息学一起，构成了一个强大的程序，用于发现细菌谱中的细微变化，并在细菌种群与健康标志物之间建立胆固醇和甘油三酯浓度方面的相关性。我们的方案具有通用性，因此在研究毒性或有益效果时，可用于研究其他有潜力改变宿主微生物群组成的化合物或营养素。
