Ellis Jeremy E, Heuser Richard, Missan Dara S, Martinez Delyn, Heningburg Avory, Shabilla Matthew, Schwartz Renata, Fry Stephen
Fry Laboratories, LLC, Scottsdale, AZ 85260, USA.
St. Luke's Phoenix Heart Center, Phoenix, AZ 85006, USA; University of Arizona, College of Medicine, Phoenix, AZ 85006, USA.
Mol Cell Probes. 2017 Jun;33:65-77. doi: 10.1016/j.mcp.2017.04.001. Epub 2017 Apr 12.
Microbial communities have been implicated in a variety of disease processes and have been intermittently observed in arterial disease; however, no comprehensive unbiased community analysis has been performed. We hypothesize that complex microbial communities may be involved in chronic vascular diseases as well and may be effectively characterized by molecular assays.
The main objective is to survey vascular debris, atheroma, and vascular filters for polymicrobial communities consisting of prokaryotic and eukaryotic microbes, specifically eukaryotic microbes.
We examined vascular aspirates of atheromatous debris or embolic protection filters in addition to matched peripheral blood samples, from fifteen patients, as well as three cadaveric coronary arteries from two separate patients, for microbial communities. General fluorescence microscopy by Höechst and ethidium bromide DNA stains, prokaryotic and eukaryotic community analysis by Next Generation DNA Sequencing (NGS), and a eukaryotic microbial 9 probe multiplexed quantitative PCR were used to detect and characterize the presence of putative polymicrobial communities. No prokaryotes were detected in peripheral blood; however, in 4 of 9 sequenced filters and in 2 of 7 sequenced atheroma debris samples, prokaryotic populations were identified. By DNA sequencing, eukaryotic microbes were detected in 4 of 15 blood samples, 5 of the 9 sequenced filters, and 3 of the 7 atheroma debris samples. The quantitative multiplex PCR detected sequences consistent with eukaryotic microbes in all 9 analyzed filter samples as well as 5 of the 7 atheroma debris samples. Microscopy reveals putative polymicrobial communities within filters and atheroma debris. The main contributing prokaryotic species in atheroma debris suggest a diverse and novel composition. Additionally, Funneliformis mosseae, an arbuscular mycorrhizal fungus in the Glomeraceae family, was detected in the coronary hard plaque from two patients. Well studied biofilm forming bacteria were not detectable in circulating peripheral blood and were not universally present in atheroma or filters. Analyses of the sequenced eukaryotes are consistent with a diverse of array poorly studied environmental eukaryotes. In summary, out of 15 patients, 6 exhibited molecular evidence of prokaryotes and 14 had molecular evidence of eukaryotic and/or polymicrobial communities in vivo, while 2 post-mortem coronary plaque samples displayed evidence of fungi.
Prokaryotes are not consistently observed in atheroma debris or filter samples; however, detection of protozoa and fungi in these samples suggests that they may play a role in arterial vascular disease or atheroma formation.
微生物群落已被认为与多种疾病过程有关，并且在动脉疾病中也有间歇性观察到；然而，尚未进行全面的无偏群落分析。我们假设复杂的微生物群落可能也参与慢性血管疾病，并且可以通过分子检测有效地进行表征。
主要目的是检测血管碎片、动脉粥样硬化斑块和血管滤器中由原核和真核微生物，特别是真核微生物组成的多微生物群落。
我们检查了15名患者的动脉粥样硬化碎片或栓子保护滤器的血管吸出物以及匹配的外周血样本，以及来自两名不同患者的三条尸体冠状动脉中的微生物群落。使用Höechst和溴化乙锭DNA染色进行一般荧光显微镜检查，通过下一代DNA测序（NGS）进行原核和真核群落分析，以及真核微生物9探针多重定量PCR来检测和表征假定的多微生物群落的存在。在外周血中未检测到原核生物；然而，在9个测序滤器中的4个以及7个测序的动脉粥样硬化斑块碎片样本中的2个中，鉴定出了原核生物种群。通过DNA测序，在15份血液样本中的4份、9个测序滤器中的5份以及7个动脉粥样硬化斑块碎片样本中的3份中检测到了真核微生物。定量多重PCR在所有9个分析的滤器样本以及7个动脉粥样硬化斑块碎片样本中的5个中检测到了与真核微生物一致的序列。显微镜检查揭示了滤器和动脉粥样硬化斑块碎片中存在假定的多微生物群落。动脉粥样硬化斑块碎片中的主要原核生物种类表明其组成多样且新颖。此外，在两名患者的冠状动脉硬斑中检测到了球囊菌科的丛枝菌根真菌摩西斗管囊霉。在循环外周血中未检测到经过充分研究的形成生物膜的细菌，并且它们在动脉粥样硬化斑块或滤器中也并非普遍存在。对测序的真核生物的分析与一系列研究较少的环境真核生物的多样性一致。总之，在15名患者中，6名在体内表现出原核生物的分子证据，14名有真核和/或多微生物群落的分子证据，而2份死后冠状动脉斑块样本显示出真菌的证据。
在动脉粥样硬化斑块碎片或滤器样本中并非始终能观察到原核生物；然而，在这些样本中检测到原生动物和真菌表明它们可能在动脉血管疾病或动脉粥样硬化斑块形成中起作用。
