Vartoukian Sonia R, Adamowska Aleksandra, Lawlor Megan, Moazzez Rebecca, Dewhirst Floyd E, Wade William G
Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
King's College London Dental Institute, London, United Kingdom.
PLoS One. 2016 Jan 14;11(1):e0146926. doi: 10.1371/journal.pone.0146926. eCollection 2016.
Over a third of oral bacteria are as-yet-uncultivated in-vitro. Siderophores have been previously shown to enable in-vitro growth of previously uncultivated bacteria. The objective of this study was to cultivate novel oral bacteria in siderophore-supplemented culture media. Various compounds with siderophore activity, including pyoverdines-Fe-complex, desferricoprogen and salicylic acid, were found to stimulate the growth of difficult-to-culture strains Prevotella sp. HOT-376 and Fretibacterium fastidiosum. Furthermore, pyrosequencing analysis demonstrated increased proportions of the as-yet-uncultivated phylotypes Dialister sp. HOT-119 and Megasphaera sp. HOT-123 on mixed culture plates supplemented with siderophores. Therefore a culture model was developed, which incorporated 15 μg siderophore (pyoverdines-Fe-complex or desferricoprogen) or 150 μl neat subgingival-plaque suspension into a central well on agar plates that were inoculated with heavily-diluted subgingival-plaque samples from subjects with periodontitis. Colonies showing satellitism were passaged onto fresh plates in co-culture with selected helper strains. Five novel strains, representatives of three previously-uncultivated taxa (Anaerolineae bacterium HOT-439, the first oral taxon from the Chloroflexi phylum to have been cultivated; Bacteroidetes bacterium HOT-365; and Peptostreptococcaceae bacterium HOT-091) were successfully isolated. All novel isolates required helper strains for growth, implying dependence on a biofilm lifestyle. Their characterisation will further our understanding of the human oral microbiome.
超过三分之一的口腔细菌尚未在体外培养成功。此前已有研究表明，铁载体能够促进此前未培养细菌的体外生长。本研究的目的是在添加铁载体的培养基中培养新型口腔细菌。研究发现，多种具有铁载体活性的化合物，包括绿脓菌素 - 铁复合物、去铁铁载体和水杨酸，能够刺激难培养菌株普雷沃氏菌属HOT - 376和苛求杆菌的生长。此外，焦磷酸测序分析表明，在添加铁载体的混合培养平板上，尚未培养的菌型戴氏菌属HOT - 119和巨球形菌属HOT - 123的比例有所增加。因此，开发了一种培养模型，将15μg铁载体（绿脓菌素 - 铁复合物或去铁铁载体）或150μl纯龈下菌斑悬液加入到琼脂平板的中央孔中，这些平板接种了来自牙周炎患者的高度稀释龈下菌斑样本。表现出卫星现象的菌落与选定的辅助菌株共同培养，转接至新鲜平板上。成功分离出五株新型菌株，它们代表了三个此前未培养的分类单元（厌氧绳菌属细菌HOT - 439，首个从绿弯菌门培养出的口腔分类单元；拟杆菌属细菌HOT - 365；以及消化链球菌科细菌HOT - 091）。所有新型分离株的生长都需要辅助菌株，这意味着它们依赖生物膜生活方式。对它们的特性分析将加深我们对人类口腔微生物组的理解。
