Panaiotov Stefan, Hodzhev Yordan, Tsafarova Borislava, Tolchkov Vladimir, Kalfin Reni
National Center of Infectious and Parasitic Diseases, 1504 Sofia, Bulgaria.
Institute of Neurobiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Microorganisms. 2021 Jul 8;9(7):1464. doi: 10.3390/microorganisms9071464.
Next-generation sequencing (NGS) and metagenomics revolutionized our capacity for analysis and identification of the microbial communities in complex samples. The existence of a blood microbiome in healthy individuals has been confirmed by sequencing, but some researchers suspect that this is a cell-free circulating DNA in blood, while others have had isolated a limited number of bacterial and fungal species by culture. It is not clear what part of the blood microbiota could be resuscitated and cultured. Here, we quantitatively measured the culturable part of blood microbiota of healthy individuals by testing a medium supplemented with a high concentration of vitamin K (1 mg/mL) and culturing at 43 °C for 24 h. We applied targeted sequencing of 16S rDNA and internal transcribed spacer (ITS) markers on cultured and non-cultured blood samples from 28 healthy individuals. Dominant bacterial phyla among non-cultured samples were Proteobacteria 92.97%, Firmicutes 2.18%, Actinobacteria 1.74% and Planctomycetes 1.55%, while among cultured samples Proteobacteria were 47.83%, Firmicutes 25.85%, Actinobacteria 16.42%, Bacteroidetes 3.48%, Cyanobacteria 2.74%, and Fusobacteria 1.53%. Fungi phyla Basidiomycota, Ascomycota, and unidentified fungi were 65.08%, 17.72%, and 17.2% respectively among non-cultured samples, while among cultured samples they were 58.08%, 21.72%, and 20.2% respectively. In cultured and non-cultured samples we identified 241 OTUs belonging to 40 bacterial orders comprising 66 families and 105 genera. Fungal biodiversity accounted for 272 OTUs distributed in 61 orders, 105 families, and 133 genera. Bacterial orders that remained non-cultured, compared to blood microbiota isolated from fresh blood collection, were Sphingomonadales, Rhizobiales, and Rhodospirillales. Species of orders Bacillales, Lactobacillales, and Corynebacteriales showed the best cultivability. Fungi orders Tremellales, Polyporales, and Filobasidiales were mostly unculturable. Species of fungi orders Pleosporales, Saccharomycetales, and Helotiales were among the culturable ones. In this study, we quantified the capacity of a specific medium applied for culturing of blood microbiota in healthy individuals. Other culturing conditions and media should be tested for optimization and better characterization of blood microbiota in healthy and diseased individuals.
新一代测序（NGS）和宏基因组学彻底改变了我们分析和鉴定复杂样本中微生物群落的能力。通过测序已证实健康个体中存在血液微生物组，但一些研究人员怀疑这是血液中的无细胞循环DNA，而另一些人则通过培养分离出了数量有限的细菌和真菌物种。尚不清楚血液微生物群的哪些部分可以复苏并培养。在此，我们通过测试添加高浓度维生素K（1mg/mL）的培养基并在43°C培养24小时，定量测定了健康个体血液微生物群的可培养部分。我们对来自28名健康个体的培养和未培养血液样本应用了16S rDNA和内部转录间隔区（ITS）标记的靶向测序。未培养样本中的优势细菌门为变形菌门（92.97%）、厚壁菌门（2.18%）、放线菌门（1.74%）和浮霉菌门（1.55%），而培养样本中变形菌门为47.83%、厚壁菌门为25.85%、放线菌门为16.42%、拟杆菌门为3.48%、蓝细菌门为2.74%、梭杆菌门为1.53%。未培养样本中真菌门担子菌门、子囊菌门和未鉴定真菌分别为65.08%、17.72%和17.2%，而培养样本中分别为58.08%、21.72%和20.2%。在培养和未培养样本中，我们鉴定出属于40个细菌目、66个科和105个属的241个操作分类单元（OTU）。真菌生物多样性占272个OTU，分布在61个目、105个科和133个属中。与从新鲜血液样本中分离的血液微生物群相比，未培养的细菌目有鞘脂单胞菌目、根瘤菌目和红螺菌目。芽孢杆菌目、乳杆菌目和棒杆菌目的物种显示出最佳的可培养性。银耳目、多孔菌目和丝孢酵母目的真菌大多不可培养。格孢腔菌目、酵母目和锤舌菌目的真菌物种属于可培养的物种。在本研究中，我们定量了用于培养健康个体血液微生物群的特定培养基的能力。应测试其他培养条件和培养基，以优化并更好地表征健康和患病个体的血液微生物群。
