体外肠道宿主-微生物界面合成细菌联合的可行性评价
Summary
采用综合口服群落、体外胃肠消化和小肠上皮模型相结合的新方法对肠道宿主-微生物相互作用进行评价。我们提出了一种方法, 可以适应评估细胞入侵的病原体和多物种生物膜, 甚至测试益生菌制剂的生存性。
Abstract
宿主和微生物群之间的相互作用已被长期承认和广泛描述。口腔与胃肠道的其他部分相似, 因为居民的微生物群发生, 防止外源细菌的定植。事实上, 在口腔中发现了600多种细菌, 单个个体在任何时候都可能携带100左右。口腔细菌具有在口腔生态系统中坚持各种利基的能力, 从而在居民微生物群落中得到整合, 有利于生长和生存。然而, 在吞咽过程中细菌进入肠道的流动已经被提出来扰乱肠道微生物群的平衡。事实上, 口服菌转移了回肠菌群中的细菌组成。我们利用合成社区作为自然口腔生态系统的简化表示, 以阐明在模拟胃肠道条件下口腔细菌的存活和生存能力。选择十四种, 经体外唾液、胃和肠道消化过程, 并提出一个 multicompartment 细胞模型, 包含 Caco-2 和 HT29-MTX 细胞模拟肠道黏膜上皮。该模型有助于解开吞噬细菌对肝肠循环细胞的影响。使用合成群落可以进行可控性和重现。因此, 这种方法可以适应评估病原体的生存能力和随后的炎症相关变化, 益生菌混合物的殖民化容量, 最终, 潜在的细菌对 presystemic 循环的影响。
Introduction
人与细菌同居, 是存在与人细胞1的同一个数字。因此, 对人类微生物群的全面了解是至关重要的。口腔是一个独特的环境, 因为它被分成几个较小的栖息地, 从而包含了大量的细菌和生物膜在这些不同的地方。作为一个开放的生态系统, 一些物种在口中可能是短暂的访客。然而, 某些微生物在出生后不久就会殖民, 形成组织的生物膜2。这些在牙龈缝隙、龈下缝隙、舌头、粘膜表面和牙科修复体和填充物3中的牙齿表面被发现。细菌也可以作为絮凝体和浮游细胞存在于牙管腔内, 或者与坏死的牙髓组织混合或悬浮在液相中。
宿主细胞与居民微生物群之间有积极、连续的交叉谈话4。细菌在物种内部和之间进行交流, 只有很小一部分自然殖民者能够坚持组织, 而其他细菌则依附于这些主要殖民者。例如, 微生物之间的细胞结合是将次级殖民者纳入口腔生物膜的关键, 并建立相互作用的微生物细胞的复杂网络4。唾液样品中大约70% 的细菌聚集物由牙龈sp、普氏菌链球菌、 Veillonella sp 和不明Bacteroidetes组成。nucleatum是龈下生物膜中的中间体殖民者, 与晚期殖民者菌、denticola和Tannerella 连翘有牵连, 这与牙周炎5有关。此外,缓症链球菌还占据粘膜和牙科栖息地, 而血统和仙人掌则更倾向于将牙齿殖民3。因此, S. 血统是存在于下门牙和犬齿, 而放线菌对内氏已发现在上部 anteriors6。
此外, 土著微生物群在维护人类健康方面起到了2的作用。居民微生物参与免疫教育和防止病原体扩张。这种殖民抵抗的发生是因为当地的细菌可以更好地适应于附着在表面上, 并且更有效地代谢可利用的养分促进生长。虽然益生菌菌株在胃肠道中存活并保持活跃, 但仍未充分描述从胃肠道上部位置吞下的本土细菌的持久性。因此, 我们接受了一个人工群落, 代表口腔生态系统, 模拟胃肠道转运条件。用类似肠道上皮的 multicompartment 模型评估细菌细胞的生存能力。目前肠道模拟器提供了适当的重现性分析的腔内微生物群落7。然而, 细菌黏附力和寄主微生物相互作用是分开解决的, 因为结合细胞系与微生物群落是挑战8。相反, 我们提出了一个框架, 提供了潜在的机械解释的成功的殖民事件报告的肠道接口。事实上, 这个模型可以与静态肠道模型联合使用, 以评估微生物群落对宿主表面信号的影响。
Protocol
1. 菌株和文化条件 注: 综合口腔群落由口腔微生物群中常见的菌株组成3。 从美国类型文化收集 (ATCC) 获得以下菌株: Aggregatibacter actinomycetemcomitans (ATCC 43718),梭杆菌 nucleatum (ATCC 10953),牙龈菌(ATCC33277),普氏菌中间体(ATCC 25611), 变形链球菌 (ATCC 25175),链球菌远缘(ATCC 33478),放线菌</em…
Representative Results
该协议导致了一个模型的生成, 适合于阐明口腔细菌在模拟胃肠道转运条件下的生存和存活。从单个菌株的完整细胞计数约 108细胞 mL-1之前, 合成社区的创建, 而多种群的缩影包含90% 的可行细胞在社区的建立 (图 1A和1B)。根据活/死定量, 细菌的生存能力减少后, 每消化步骤 (图 2)。这可能?…
Discussion
口服微生物菌群是人类健康的一个关键因素, 最近有几个作者20、21发表了报告。先前的研究表明, 摄取含有大量细菌的唾液会影响小肠的微生物生态系统, 这是免疫启动的主要部位之一。静态上消化道消化模型与由肠道上皮和粘液分泌细胞代表的宿主界面的结合, 有助于解开微生物成分对宿主的影响。
体外模型是研究的基本工…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢佛兰德研究基金会对玛尔塔卡拉塔余德 (FWO 博士后 fellowship-12N2815N) 的财政支持。艾玛-加西亚萨纳布里亚是一个博士后研究员支持的富兰德创新和企业家精神 (Agentschap 与客厅里 Innovatie 门 Wetenschap créative, 内陆水运)。
Materials
| STRAINS | |||
| Aggregatibacter actinomycetemcomitans | American Type Culture Collection | ATCC 43718 | |
| Fusobacterium nucleatum | American Type Culture Collection | ATCC 10953 | |
| Porphyromonas gingivalis | American Type Culture Collection | ATCC 33277 | |
| Prevotella intermedia | American Type Culture Collection | ATCC 25611 | |
| Streptococcus mutans | American Type Culture Collection | ATCC 25175 | |
| Streptococcus sobrinus | American Type Culture Collection | ATCC 33478 | |
| Actinomyces viscosus | American Type Culture Collection | ATCC 15987 | |
| Streptococcus salivarius TOVE-R | |||
| Streptococcus mitis | American Type Culture Collection | ATCC 49456 | |
| Streptococcus sanguinis | BCCM/LMG Bacteria Collection | LMG 14657 | |
| Veillonella parvula | Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures | DSM 2007 | |
| Streptococcus gordonii | American Type Culture Collection | ATCC 49818 | |
| CELL LINES | |||
| Caco-2 cells | European Collection of Authenticated Cell Cultures | 86010202 | |
| HT29-MTX cells | European Collection of Authenticated Cell Cultures | 12040401 | |
| REAGENTS AND CONSUMABLES | |||
| Brain Heart Infusion (BHI) broth | Oxoid | CM1135 | |
| Blood Agar 2 | Oxoid | CM0055 | Blood Agar medium |
| Menadione | Sigma | M9429 | |
| Hemin | Sigma | H9039 | |
| 5% sterile defibrinated horse blood | E&O Laboratories Ltd, | P030 | |
| InnuPREP PCRpure Kit | Analytik Jena | 845-KS-5010250 | PCR purification kit |
| Big Dye | Applied Biosystems | 4337454 | Dye for sequencing |
| ABI Prism BigDye Terminator v3.1 cycle sequencing kit | Applied Biosystems | 4337456 | |
| SYBR Green I | Invitrogen | S7585 | |
| Propidium Iodide | Invitrogen | P1304MP | |
| T25 culture flasks uncoated, cell-culture treated, vented, sterile | VWR | 734-2311 | |
| Trypsin-EDTA solution | Sigma-Aldrich | T3924-100ML | |
| Trypan Blue solution 0.4%, liquid, sterile-filtered |
Sigma-Aldrich | T8154 | |
| PBS | Gibco | 14190250 | |
| DMEM cell culture media, with GlutaMAX and Pyruvate | Life technologies | 31966-047 | |
| Corning Transwell polyester membrane cell culture inserts | Sigma-Aldrich | CLS3450-24EA | |
| Mucin from porcine stomach Type II | Sigma-Aldrich | M2378 | |
| Inactivated fetal bovine serum | Greiner Bio One | 758093 | |
| Antibiotic-Antimycotic (100X) | Gibco | 15240062 | |
| Triton X 100 for molecular biology | Sigma-Aldrich | T8787 | |
| DPBS without calcium, magnesium | Gibco | 14190-250 | |
| Pierce LDH Cytotoxicity Assay Kit | Thermo Fisher Scientific | 88953 | |
| Corning HTS Transwell-24 well, pore size 0.4 µm | Corning Costar Corp | 3450 | |
| Nuclease-free water | Serva Electrophoresis | 28539010 | |
| EQUIPMENT | |||
| Neubauer counting chamber improved | Carl Roth | T729.1 | |
| BD Accuri C6 Flow cytometer | BD Biosciences | 653118 | |
| PowerLyzer 24 Homogenizer | MoBio | 13155 | |
| T100 Thermal Cycler | BioRad | 186-1096 | |
| Flush system | Custom made | – | |
| InnOva 4080 Incubator Shaker | New Brunswick Scientific | 8261-30-1007 | Shaker for 2.10 |
| Memmert CO2 incubator | Memmert GmbH & Co. | ICO150med | |
| Millicell ERS (Electrical Resistance System) | EMD Millipore, Merck KGaA | MERS00002 | |
| Millipore Milli-Q academic, ultra pure water system | Millipore, Merck KGaA | – | |
| Shaker (ROCKER 3D basic) | IKA | 4000000 | Shaker for 6.10 |
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Cite This Article
Calatayud Arroyo, M., Van de Wiele, T., Hernandez-Sanabria, E. Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface. J. Vis. Exp. (137), e57699, doi:10.3791/57699 (2018).