基于高通量测序和培养组学的血小板中细菌检测方法的建立

Establishment of detection method for bacteria in platelets based on high-throughput sequencing and culturomics

目的建立1种基于高通量测序培养组学的细菌检测体系,以此来提高血小板中细菌的检出率。方法利用微生物高通量培养组细菌检测体系,配置培养组中的8种液体培养基,即血培养瓶(37℃)、含5 mL羊血的血培养瓶(37℃)、100 mL海生菌肉汤(37℃)、含瘤胃液和羊血各5 mL的血培养瓶(37℃)、100 mL胰蛋白大豆肉汤(37℃)、100 mL5%羊血肉汤(28、37℃)、含5 mL胃液血培养瓶(37℃)和替代传统的1种培养基-血培养瓶(37℃),取血小板标本9份(5 mL/份)做预培养:在恒温有氧条件下的液体培养基中培养60 d,将液体培养基中的培养液9份,在培养24 h后第1次用无菌注射器吸取100μL菌到血琼脂平板上,并用涂布棒涂布均匀;此后每2—3 d重复同样操作1次。血琼脂平板与预培养在温度相同的条件下培养1—2 d,用挑菌环挑取血平板上的单个菌株,用DNA Blood Mini Kit提取DNA后,采用通用引物27F/1429R扩增该细菌的16S rRNA基因片段并采用Sanger测序鉴定菌种,同时将各血小板标本混匀后分别取40 mL以96800 g离心4 h,弃上清;采用DNA Blood Mini Kit提取沉淀总DNA,构建DNA文库后在Illumina Illumina HiSeq 4500上深度测序,所得数据用自有分析软件KrakenPy 1.0分析其中的微生物组并鉴定细菌种类。将宏基因组高通量测序方法得到的结果和培养组的结果相互验证,确认致血小板污染的细菌种类。结果经过细菌培养组培养后分离单个菌落,用Sanger测序鉴定出的细菌有:赖氨酸芽孢杆菌(Lysinibacillus sp.)、芽孢杆菌(Bacillus sp.)、郭霍氏芽孢杆菌(B.kochii)、蜡样芽孢杆菌(B.cereus)、类芽孢杆菌(Paenibacillus sp.)、黄原胶降解菌(P.lautus)、葡萄球菌(Staphylococcus sp.)、马葡萄球菌(S.equorum)、红球菌(Rhodococcus sp.)。宏基因组测序产出Raw data共72.22 G,显示丰度较高的主要为厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、变形菌门(Proteobacteria),其中鉴定出细菌种类最多的厚壁菌门(Firmicutes)细菌种类:苏云金芽孢杆菌(B.thuringiensis)、蜡状芽孢杆菌(B.cereus)、类芽孢杆菌(Paenibacillus sp.)、胶质类芽孢杆菌(P.mucilaginosus);其次为变形菌门(Proteobacteria)的细菌主要包括:赭黄嗜盐囊菌(Haliangiumochraceum)、嗜麦芽寡养单胞菌(S.maltophilia);还有放线菌门(Actinobacteria),主要包括红球菌(Rhodococcus sp.)、红平红球菌(R.erythropolis)、银色棒杆菌(Corynebacteriumargentoratense)。培养组学方法与宏基因组测序2种方法都检测出的细菌:蜡样芽孢杆菌(B.cereus)、类芽胞杆菌(Paenibacillus sp.)、红球菌(Rhodococcus sp.)。结论高通量微生物培养组学方法培养出了高丰度的蜡样芽孢杆菌(B.cereus)、类芽孢杆菌(Paenibacillus sp.)和红球菌(Rhodococcus sp.)等最可能污染血小板标本的细菌,但未培养出变形菌门(Proteobacteria)、放线菌门(Actinobacteria)等血小板中可能存在的细菌。高通量培养组学方法与宏基因组测序方法相结合为高灵敏地检测血小板中的细菌提供了新的思路。

Objective To establish a bacterial detection system based on high-throughput sequencing culturomics to improve the detection rate of bacteria in platelets.Methods Using the bacterial detection system of microorganism highthroughput culture group,8 kinds of liquid culture media in the culture group were configured,namely blood culture bottle(37℃),blood culture bottle containing 5mL sheep blood(37℃),marine bacteria broth(37℃),blood culture bottle containing rumen fluid and sheep blood(37℃),pancreas soybean broth(37℃),sheep blood broth(28℃,37℃),and blood culture bottle containing rumen fluid(37℃)as the substitute of the traditional medium-blood culture bottle(37℃).9 platelet samples were precultured,i.e.cultured in liquid culture medium at 37℃or 28℃under constant temperature and aerobic condition for 60 days.Every kinds of culture liquid in liquid culture medium were sucked into blood agar plate with sterile syringe after the first 24h,and coated evenly with coating rod.The same operation was performed every 2—3 days thereafter.Blood agar plate and preculture were cultured at the same temperature for 1—2 days.Single strain on the blood plate was picked out by using a pick ring.After DNA was extracted by DNA Blood Mini Kit,16S rRNA gene fragment of the bacterium was amplified by universal primer 27F/1429R and the strain was identified by Sanger sequencing.At the same time,every platelet samples were mixed and centrifuged at 40mL at 96800 g for 4 hours,and the supernatant was discarded.The precipitated total DNA was extracted by DNA Blood Mini Kit,and the DNA library was constructed and sequenced deeply via Illumina Illumina HiSeq 4500.The data obtained was analyzed by its own analysis software named KrakenPy 1.0 and the bacterial species were identified.The results obtained by the metagenomic high-throughput sequencing method and the results of the culture group were mutually verified to confirm the bacterial species causing platelet contamination.Results A single colony after culture was separated by microbial culturomics,and the bacteria identified by Sanger sequencing were:Lysinibacillus sp.,Bacillus sp.,Bacillus Koch,Bacillus cereus,Paenibacillus sp.,P.lautus,Staphylococcus sp.,S.equorum and Rhodococcus sp..The metagenome sequencing produced a total of 72.22 G of Raw data,showing that the high abundance was mainly found in Firmicutes,Actinobacteria and Proteobacteria.Among them,the bacteria species of Firmicutes with the most bacteria species were identified:B.thuringiensis,B.cereus,Paenibacillus sp.and P.mucilaginosus.Secondly,the bacteria of Proteobacteria mainly included:Haematitum ochraceum and Stenotrophomonas maltophilia;there were also Actinobacteria,mainly including Rhodococcus sp.,R.erythropolis and Corynebacterium argentoratense.Bacteria detected by both culturomics and metagenomics sequencing method were:B.cereus,Paenibacillus sp.and Rhodococcus sp..Conclusion High-throughput microbiological culturomics has cultured the bacteria with high abundance,such as B.cereus,Paenibacillus sp.and Rhodococcus sp.,which are most likely to contaminate platelet specimens,but the bacteria that may already exist in platelets has not been cultured yet,such as Proteobacteria and Actinobacteria.The combination of high throughput culturomics and metagenome sequencing provides new approaches for highly sensitive detection of bacteria in platelets.