The Healthy Human Blood Microbiome: Fact or Fiction?

Author: Diego J. Castillo1, Riaan F. Rifkin1,2, Don A. Cowan1 and Marnie Potgieter 1
Affiliation: 1 Department of Biochemistry, Genetics and Microbiology, Centre for Microbial Ecology and Genomics, University of Pretoria,Pretoria, South Africa,
2 Human Origins and Palaeo Environmental Research Group, Department of Anthropology and Geography, Oxford Brookes University, Oxford, United Kingdom

  这是一篇讨论人类血液微生物组综述！

  人们以往的认知里，血液中发现的microbes一贯地被当作发生感染的迹象，然而，在健康人群中血液中存在微生物组( “healthy” human blood microbiome ,HBM)的证据正逐渐累积。
  随着NGS以及WMGS等技术的应用，人们进行大规模的genetic以及方面的研究，其中就包括 “Metagenomes of the Human Intestinal Tract” project和 “Human Microbiome Project” (HMP)，两项研究包含2000以上的参与者，为建立人类健康个体的微生物结构的建立起到了至关重要的作用。尽管当前大多的研究都集中在肠道微生物，但是其他方面的研究，包括皮肤、口腔、眼、肺、胎盘以及尿道的微生物组研究也同样存在。
  Human blood comprises ∼54.3% plasma, ∼45% red blood cells (erythrocytes), ∼0.7% white blood cells (lymphocytes)and a variable number of platelets (thrombocytes), depending on health status. Blood is the liquid medium that carries and sustains the most basic, but most essential, elements of life. Whereas erythrocytes are responsible primarily for the transport of oxygen, lymphocytes serve as a highly efficient surveillance system that monitors the blood for invasive microbes.The primary function of thrombocytes is to react to bleeding from blood vessel injury by clotting. Because blood has traditionally been considered to be a sterile environment, devoid of all other forms of foreign (e.g., bacterial) cells, it is not surprising that the concept of a healthy HBM has been met with criticism.
  人类HBM研究时间轴如下:

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CONTROVERSY AND EVOLUTION OF A NOVEL CONCEPT

  最早提出HBM并引发争议的研究可追溯到1960s 。Tedeschi1发表了在健康人体血液中存在代谢活跃的细菌相关文献。特别是他们提出一种假设，由于红细胞悬液对核酸和氨基酸的吸附增加使支原体样(mycoplasm-like) or L型(L-phase ,cell-wall deficient) 细菌能够在非疾病的个体的血液中出现。另外，在1977年Gerald Domingue和Jorgen Schlegel2报道约~7%的健康个体的血液样本通过裂解过滤后能够出现细菌生长。
  随着技术发展，越来越多健康个体血液中的细菌DNA3-5发现并通过相关技术( qPCR,transmission electron microscopy (TEM), dark-field microscopy (DFM), fluorescence in situ hybridization (FISH) and the sequencing of PCR-amplified 16S rRNA)被证实。
  对正常情况下健康人的血液是无菌的传统概念的挑战引起了学术界相当大的争议。Mitchell6对McLaughlinDNA3等人和其他研究的进行审查后得出结论，在健康人类血液中发现的多形性细菌不过是来自分解的红细胞碎片。Martel7的研究发现bacteria-like structures与resembled membrane vesicles非常相似，并且被暗视野显微镜捕获的振动折射粒子只是血液蛋白的聚集物，这些证据支持了Mitchell6的观点。尽管血液中微生物的视觉检测还需要进一步确认，但是微生物的遗传物质在血液循环系统中的富集已经得到了广泛的研究证实。
  为了挑战人类血液的“germ-free”概念，方法学上的障碍阻碍了对HBM的研究。自然条件下，在人类血液中发现的微生物实际上处于休眠状态(dormant state)8，所以，培养不能被引入来作为支持存在HBM的方法。另外，mb DNA(microbe DNA derive from blood)的浓度都非常低，主要是应用qPCR以及Targeted NGS对血液中 “innocuous” 细菌进行检测。
  low-biomass的微生物组容易被是外源性污染所影响，因为在DNA提取以及文库制备时大约会受到>90 种微生物属的影响，这些污染来自试剂和实验室环境，它们均会对基于序列的HBM分析的产生严重的影响9-10。
  接下来的实验将会更侧重于研究在健康个体中存在的细菌是living or dead，激活亦或非激活的微生物菌属。文章提出了检测微生物活性的可靠的方法:propidium monoazide (PMA) treatment and cellular energy measurements11.另外，除了细菌，archaeal12、 fungi13、viral14等DNA物质还很少被报道。Moustafa14提到19种健康个体的血液病毒组分类。另外，还有被确认在健康个体中出现的真核病毒，如： rhabdoviruses、 anelloviruses，以及其他家族类别：Herpesviridae 以及Poxiviridae。但这些确认出现的病毒还需要进一步确认是resident members of the HBM或者是由于偶然感染后进入机体的。

ORIGIN AND LOCATION OF BLOOD-BORNE BACTERIA

   在生态位占有一席之地亦或仅仅是单纯transient residents也是争论的焦点。

  • 焦点1 常见的易位解剖位：gastro-intestinal tract、skin以及口腔。Whittle15通过将HMP microbiome data和 HBM数据进行比较，发现HBM更接近于皮肤和口腔的微生物组结构，与肠道微生物存在更大差异。微生物肠道易位方式包括通过DC吸附后通过肠道上皮细胞或肠道分泌粘液的杯状细胞；另外M细胞也能参与肠道细菌向血液循环易位的过程。
  • 焦点2 vertical transmission垂直传播
  • 焦点3 因为mb的起源相当复杂，且易受各种环境因素以及自身免疫系统的调节，据此推论如果各种mb的起源确实来自身体其他部位，而且这些易位的发生不是频繁事件，那么健康个体的HBM是高度动态变化的。但，近期的多项研究报道HBM为较为稳定的，主要的mb包括16-24：Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes。
  • 焦点4 HBM的定位：erythrocytes与leukocytes均可观察到。已有文献报道的正常人外周血中微生物，肺炎支原体(Chlamydia pneumoniae)25，Staphylococcus aureus26-27.另外，Païssé21提出，93.74%的细菌DNA由WBC或PLT包裹的状态(buffy coat ,BC)。相似的，某些细菌可以直接进入RBCs，并且持续存在于营养丰富的RBCs中。比如，Staphylococcus aureus28利用铁做为营养来源存在于RBCs中，相似的还有Streptococcus pneumoniae、Brucella melitensis、Francisella tularensis。
COMPOSITION OF THE PUTATIVE HEALTHY HUMAN BLOOD-MICROBIOME

   HBM 最主要的门类是 Proteobacteria 随后是 Actinobacteria, Firmicutes以及 Bacteroidetes。HBM具有更为易变且复杂的多样性。Moriyama29的研究显示HBM的组成为Bacillus, Flavobacteria, Stenotrophomas 以及Serratia.基于培养，Damgaard30的研究显示62%健康个体血液能够观察到微生物生长，最主要的为Propionibacterium acnes
以及Staphylococcus epidermis,同时也存在 Bacilli 和 Micrococcus species。Païssé21分析了血液中不同成分的细菌组成，纲水平上在RBCs中Fusobacteria 以及Flavobacteria的含量最高，其中，目水平上，血浆与erythrocyte中的Clostridia 含量最高。7种属水平的细菌在RBCs中被鉴别到，分别为两种条件致病菌，Acinetobacter baumanni 和
Stenotrophomonas maltophilia.这些结果可能能够真实的反应在血液中的确存在相应的微生物，但同样也存在一定问题，因为Glassing31对DNA提取试剂的污染DNA来源研究显示，最主要的污染菌包括了:Bacillus, Flavobacteria, Fusobacteria, Propionibacterium以及 Serratia。

THE CLINICAL RELEVANCE OF THE HEALTHY HBM
CONCLUDING REMARKS AND FUTURE PROSPECTS
文献
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