研究目的：

本研究旨在通过微生物培养组学（Culturomics）培养血小板中存在且常规培养方法不易培养的细菌，探究血小板中可培养细菌的情况，并通过宏基因组二代测序（Metagenomics Next-generation Sequencing，mNGS）作为验证，两种方法结合进行探究了解血小板中细菌组成，从而提高血小板中存在的细菌的检出率，为血小板细菌筛查检测提供新思路，进而保障血液安全。

研究方法：

（1）血小板细菌长周期培养检测方法策略研究：收集6例健康人的血小板标本，配制8种置于需氧环境中的培养基和12种置于厌氧环境中的培养基对血小板标本中的细菌进行预培养，血小板接种至需氧培养基24h和厌氧培养基48h后，将液体培养基涂布至血琼脂平板上分离单菌落，每三天重复此操作步骤，直至30天结束。对分离培养获得的单克隆菌株进行16S rRNA测序鉴定分析确定单菌落的种属。接种至液体培养基的血小板经30天（D30）培养结束后取样，对取样标本和血小板原液进行核酸提取、DNA建库、文库质检后采用宏基因组测序技术进行测序，测序数据进行比对分析血小板标本中的细菌种属和丰度。宏基因组测序结果和培养组结果相互验证，确认血小板中存在以及可培养的细菌种类。

（2）血小板细菌短周期培养检测方法策略研究：收集2例健康人的血小板标本，同样将血小板接种至需要置于需氧环境中的8种培养基和置于厌氧环境中的12种培养基中进行预培养，之后定期将液体培养基涂布至血琼脂平板上进行分离培养直至10天结束。对分离培养获得的单克隆进行16S rRNA测序鉴定分析确定单菌落的种属。同时，对血小板原液采用宏基因组测序技术进行测序，宏基因组测序结果和培养组结果相互验证确定血小板标本中的细菌种类，并与长周期培养检测结果进行对比。血小板接种至液体培养基培养的第3天（D3）、第5天（D5）和第10天（D10）分别取样，对取样标本采用宏基因组测序技术进行测序，测序数据进行比对分析液体培养基中细菌的种属和丰度，分析取样三天不同培养基丰度占优势的菌种的差别。同时对丰度较高的致病菌及机会致病菌进行数据分析，确定液体培养基对致病菌及机会致病菌扩大培养的效果。

结果：

（1）长周期培养的血小板标本经定期转移至血琼脂平板培养长出单克隆后进行分离鉴定，共分离获得90株细菌，隶属于3个门，5个纲，5个目，7个科，9个属，23个种，其中在种水平上得到的单克隆较多的菌株为aurantiaca短波单胞菌(Brevundimonas aurantiaca)(16.7%)，Bacillus sp. Y1(15.6%)，痤疮丙酸杆菌（Cutibacterium acnes）(14.4%）和短短芽孢杆菌（Brevibacillus brevis）(13.3%)；短周期培养的血小板标本仅分离出2种细菌，分别是痤疮丙酸杆菌（C. acnes）和蜡样芽孢杆菌（B. cereus）。

（2）长周期培养的血小板原液经Illumina NovaSeq 6000测序后分析发现变形菌门（Proteobacteria），厚壁菌门（Firmicutes）和放线菌门（Actinobacteria）丰度值较高，培养组学方法与宏基因组测序两种方法都检测出的细菌为：厚壁菌门细菌种类：B. sp. Y1、苏云金芽孢杆菌（B. thuringiensis）、蜡样芽孢杆菌（B. cereus）、运动芽孢杆菌（B. mobilis）、贝莱斯芽孢杆菌（B. velezensis）、短短芽孢杆菌（B. brevis）和表皮葡萄球菌（Staphylococcus epidermidis）；放线菌门细菌种类：痤疮丙酸杆菌（C.acnes）；变形菌门细菌种类：大肠埃希菌（Escherichia coli）和戴尔福特菌（Delftia tsuruhatensis）。短周期培养的血小板原液经测序后发现变形菌门（Proteobacteria）在两个标本中的丰度值较高，分析D3、D5和D10的液体培养基取样标本的测序数据，我们发现不同培养基不同取样时间排名前十的优势菌种在细菌种类上基本能保持一致，但有些菌种随着培养时间丰度上升，有些菌种随着培养时间丰度下降。我们也分析了不同培养基不同取样时间的优势菌种，发现这些优势菌种大多为不致病的细菌，少数为机会致病细菌，极少数则为致病细菌。对丰度较高的致病菌及机会致病菌进行分析，发现液体培养基确实可以扩大培养血小板保存期内存在的细菌，细菌丰度明显上升，且不同培养基可以针对性扩大培养不同菌种。

（3）短周期培养与长周期培养相比，短周期培养分离出的单菌落数目及种类明显减少。长周期培养和短周期培养通过培养组学分离得到的大多数单菌落在宏基因组测序数据中能够得到验证，也有部分单菌落并未得到验证，宏基因组测序分析丰度较高的一些菌种经培养组学培养未得到其单菌落，且大多数优势菌种的来源也未可知，仍需探索。

结论：

在长周期培养中，培养组学和宏基因组学方法相互验证培养鉴定出一些细菌，证明血小板中存在可培养的细菌，初步证明了该检测方法的可行性。缩短培养周期，分离得到的单菌落数目和种类有所下降，说明培养组学在短时间的细菌检测方面仍有缺陷，宏基因组学检测到的优势菌种证明了液体培养基在血小板保存期内可扩大培养血小板中存在的细菌，进一步证明培养组学与宏基因组学相结合的检测技术确是可行的。但大多数优势菌种尚未在血小板及制品污染中报道，来源并不清楚，仍需进一步探索。

Background and Objectives:

The primary aim of this study is to explore the novel approach to culturing bacteria in platelets by culturomics, which were previously difficult to culture using conventional methods. Subsequently, we validated the efficacy of this method using Metagenomics Next-generation Sequencing (mNGS). Through the combination of culturomics and mNGS, we investigated the bacterial compositions within platelets. Our study in combined method contributes to improving the detection rate of bacteria in platelets, providing new insights into platelet bacterial screening and detection, thereby enhancing blood safety measures.

Methods:  

Long-term culture and detection for platelet bacteria: platelet samples from 6 healthy people were collected for the experiment. 8 kinds of culture medium placed in an aerobic environment and 12 kinds of culture medium placed in the anaerobic environment were prepared to pre-culture bacteria in platelet samples. After platelets were cultured in the aerobic medium for 24h and anaerobic medium for 48h, the liquid medium was coated to separate single colonies on a blood agar plate, and the operation steps were repeated every 3 days until the end of 30 days. 16S rRNA sequencing was used to identify the species of single colony. Platelets inoculated into liquid medium were sampled after 30 days (D30) of culture. After nucleic acid extraction, DNA library construction, and library quality inspection, the samples and platelet stock solution were sequenced by metagenomic sequencing technology. The sequencing data were compared and analyzed for bacterial species and abundance in platelet samples. The results of metagenomics and culturomics were mutually verified to confirm the species of culturable bacteria in platelets.

(2) Short-term culture and detection for platelet bacteria: platelet samples from 2 healthy people were collected, and the platelets were also inoculated into 8 kinds of culture medium that need to be placed in an aerobic environment and 12 kinds of culture medium that need to be placed in anaerobic environment for pre-culture. After that, the liquid medium was regularly coated on the blood agar plate for separation and culture until the end of 10 days. 16S rRNA sequencing was used to identify the species of a single colony. At the same time, the platelet stock solution was sequenced by metagenomic sequencing technology. The results of metagenomics and culturomics were mutually verified to determine the bacterial species in platelet samples, and compared with the results of long-term culture. Samples were taken on the 3rd day (D3), 5th day (D5) and 10th day (D10) after platelets were inoculated into the liquid culture medium. The samples were sequenced by metagenomic sequencing technology. The sequencing data were compared and analyzed for the species and abundance of bacteria in the liquid culture medium, and the differences of dominant strains in different media were analyzed. At the same time, the data of pathogenic bacteria and opportunistic pathogens with high abundance were analyzed to determine the effect of liquid medium on the expansion of pathogenic bacteria and opportunistic pathogens.

Results:

The long-term cultured platelet samples were regularly transferred to a blood agar plate for culture, and single colonies were isolated. A total of 90 strains of bacteria were isolated, belonging to 3 phyla, 5 classes, 5 orders, 7 families, 9 genera, and 23 species. The strains with more single colonies at the species level were Brevundimonas aurantiaca (16.7%), Bacillus sp. Y1 (15.6%), Cutibacterium acnes (14.4%) and Brevibacillus brevis (13.3%). Only 2 kinds of bacteria were isolated from the short-term cultured platelet samples, namely, C.acnes and B. cereus.

The long-term cultured platelet samples were sequenced by Illumina novaseq 6000 and found that the abundance values of Proteobacteria, Firmicutes, and Actinobacteria were high. The bacteria detected by both culturomics and metagenomic sequencing methods were as follows: Firmicutes: B. sp. Y1, B. thuringiensis, B. cereus, B. mobilis , B. velezensis, Staphylococcus epidermidis, and Brevibacillus brevis; Actinobacteria: C. acnes; Proteobacteria: Escherichia coli and Delftia tsuruhatensis. After sequencing of short-term cultured platelet stock solution, it was found that the abundance of Proteobacteria was higher in the two samples. By analyzing the sequencing data of liquid medium samples from D3, D5, and D10, we found that the top ten dominant bacteria in different mediums and sampling time were the same in bacterial species. However, the abundance of some strains increased with the culture time, while the abundance of some strains decreased with the culture time. We also analyzed the species of bacteria in different medium and sampling time. Our findings revealed that the majority of these dominant bacteria were non-pathogenic bacteria, with a minority being opportunistic pathogens and a small number categorized as pathogenic bacteria. By analyzing the high abundance of pathogenic bacteria and opportunistic pathogens, we found that the liquid medium could help expand the culture of bacteria during the platelet preservation period, leading to a significant increase in bacterial abundance. Moreover, different types of medium can be tailored to selectively expand the cultivation of different bacterial strains.

(3) Compared with long-term culture, the number and species of single colonies isolated from short-term culture were significantly reduced. Most single colonies isolated from both long-term culture and short-term culture via culturomics were identified in metagenomic sequencing. Metagenomic sequencing analysis showed that some strains with high abundance did not get their single colonies after culture, and the origins of most dominant strains remained unidentified, which is still worth further exploration.

Conclusions：

In the long-term culture, combined culturomics and metagenomics methods successfully identified certain bacteria, affirming the presence of culturable bacteria in platelets, which preliminarily proved the feasibility of our detection method. However, when the culture cycle was shortened, there was a decrease in the number and species of isolated single colonies, indicating the limitations in the efficacy of culturomics for short-term bacterial detection.

Additionally, the dominant bacteria detected by metagenomics proved that the liquid medium could facilitate the expansion of the bacteria in cultured platelets during the storage period of platelets, thereby reinforcing the feasibility of our combined detection technology integrating culturomics and metagenomics. However, the majority of the dominant bacteria have not been previously reported in the contamination of platelets and products, and their origin remain unclear, necessitating further investigation.