Jarocki Piotr, Podleśny Marcin, Komoń-Janczara Elwira, Kucharska Jagoda, Glibowska Agnieszka, Targoński Zdzisław
Department of Biotechnology, Human Nutrition and Food Commodities, University of Life Sciences in Lublin, 8 Skromna St., 20-704, Lublin, Poland.
BMC Microbiol. 2016 Jul 22;16(1):159. doi: 10.1186/s12866-016-0779-3.
Members of the genus Bifidobacterium are anaerobic Gram-positive Actinobacteria, which are natural inhabitants of human and animal gastrointestinal tract. Certain bifidobacteria are frequently used as food additives and probiotic pharmaceuticals, because of their various health-promoting properties. Due to the enormous demand on probiotic bacteria, manufacture of high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. Additionally, isolation of new industrial bacteria from various environments may lead to multiple isolations of the same strain, therefore, it is important to apply rapid, low-cost and effective procedures differentiating bifidobacteria at the intra-species level. The identification of new isolates using microbiological and biochemical methods is difficult, but the accurate characterization of isolated strains may be achieved using a polyphasic approach that includes classical phenotypic methods and molecular procedures. However, some of these procedures are time-consuming and cumbersome, particularly when a large group of new isolates is typed, while some other approaches may have too low discriminatory power to distinguish closely related isolates obtained from similar sources.
This work presents the evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level. Our experiments included 17 reference strains and showed that in comparison to ARDRA, genotypic fingerprinting procedures (RAPD and rep-PCR) seemed to be less reproducible, however, they allowed to differentiate the tested microorganisms even at the intra-species level. In general, RAPD and rep-PCR have similar discriminatory power, though, in some instances more than one oligonucleotide needs to be used in random amplified polymorphic DNA analysis. Moreover, the results also demonstrated a high discriminatory power of SDS-PAGE fingerprinting of whole-cell proteins. On the other hand, the protein profiles obtained were rather complex, and therefore, difficult to analyze.
Among the tested procedures, rep-PCR proved to be the most effective and reliable method allowing rapid differentiation of Bifidobacterium strains. Additionally, the use of the BOXA1R primer in the differentiation of 21 Bifidobacterium strains, newly isolated from infant feces, demonstrated slightly better discriminatory power in comparison to PCR reactions with the (GTG)5 oligonucleotide. Thus, BOX-PCR turned out to be the most appropriate and convenient molecular technique in differentiating Bifidobacterium strains at all taxonomic levels.
双歧杆菌属成员是厌氧革兰氏阳性放线菌，是人和动物胃肠道的天然定植菌。某些双歧杆菌因其具有多种促进健康的特性，常被用作食品添加剂和益生菌药物。由于对益生菌的巨大需求，生产含有活微生物的高质量产品需要快速准确地鉴定特定细菌。此外，从各种环境中分离新的工业用细菌可能导致同一菌株的多次分离，因此，应用快速、低成本且有效的程序在种内水平区分双歧杆菌很重要。使用微生物学和生化方法鉴定新分离株很困难，但使用包括经典表型方法和分子程序的多相方法可以实现对分离菌株的准确表征。然而，其中一些程序耗时且繁琐，特别是在对大量新分离株进行分型时，而其他一些方法的鉴别力可能过低，无法区分从相似来源获得的密切相关的分离株。
本研究对四种分子方法（ARDRA、RAPD-PCR、rep-PCR和SDS-PAGE指纹图谱）的鉴别力进行了评估，这些方法广泛用于双歧杆菌在菌株水平的快速区分。我们的实验包括17株参考菌株，结果表明，与ARDRA相比，基因型指纹图谱程序（RAPD和rep-PCR）的可重复性似乎较低，然而，它们甚至在种内水平也能区分受试微生物。一般来说，RAPD和rep-PCR具有相似的鉴别力，不过，在某些情况下，随机扩增多态性DNA分析需要使用不止一种寡核苷酸。此外，结果还表明全细胞蛋白的SDS-PAGE指纹图谱具有很高的鉴别力。另一方面，获得的蛋白质图谱相当复杂，因此难以分析。
在测试的程序中，rep-PCR被证明是区分双歧杆菌菌株最有效、最可靠的方法。此外，在区分从婴儿粪便中新分离的21株双歧杆菌菌株时，使用BOXA1R引物与使用(GTG)5寡核苷酸进行PCR反应相比，鉴别力略好。因此，BOX-PCR被证明是在所有分类水平区分双歧杆菌菌株最合适、最方便的分子技术。
