Establishment Of A Method For The Detection Of Pathogens In Platelets Based On High-throughput Sequencing And Culturomics

Objective:The high-throughput culturomics has made great progress in the cultivation of intestinal flora.This method can not only cultivate bacteria that can grow on the traditional culture medium,but also cultivate a variety of bacteria that were previously considered "unable to be cultivated".However,bacterial contamination in platelets has always been a problem in the field of blood collection and supply.Therefore,we plan to use the method of culturomics to carry out accurate detection of platelets and achieve the following objectives.(1)To establish a bacterial detection system by using the method of microbial high-throughput culturomics,and using this system to detect bacteria in contaminated platelets,so as to improve the detection rate of bacteria in platelets.At the same time,metagenomic high-throughput sequencing was applied to verify the above detection system.(2)To identify bacterial species cultured from inoculated samples by using 16S rRNA first generation sequencing and 16S depth sequencing;High-throughput metagenomics is used to directly identify bacterial species in platelets,and the three methods are comprehensively analyzed to finally determine the bacteria contained in platelets and analyzing the results.Methods:(1)Eight kinds of liquid culture media under aerobic conditions in culturomics are adopted to replace the traditional one,the collected platelets are inoculated into the liquid culture media for pre-culture,and every kind of culture solution in the liquid culture media is regularly and correspondingly coated on a blood agar plate,a single strain is separated and 16S rRNA gene fragments of the bacterium are amplified,and then base determination is carried out by Sanger sequencing method;The sequencing results were compared with BLAST on NCBI website to identify the bacteria species.(2)Mixing samples of liquid culture medium in preculture,washing bacteria in blood agar solid culture medium with sterile PBS solution,mixing samples,then using 16S depth sequencing of the two mixed samples respectively,analyzing sequencing results by bioinformatics method and identifying bacteria species.(3)After ultracentrifugation of platelets,the total DNA was extracted,and the DNA library was built and sequenced by Metagenomics sequencing.The data obtained were analyzed with KrakenPy 1.0 software owned by our research group and the bacterial species were identified.(4)The results of metagenome sequencing and the results of culturomics are mutually verified to determine the bacterial species causing the contaminated platelets,and the identified bacteria are analyzed.Results:(1)A culture system for detecting bacteria in platelets was established and BLAST was used to identify strains after Sanger sequencing.By this method,Bacillus sp.,Lysinibacillus sp.,B.kochii,B.cereus,Paenibacillus sp.,P.lautus,Staphylococcus sp.,S.equorum and Rhodococcus sp.were identified.(2)A total of 166,678 Raw Tags were obtained by 16S depth sequencing,161,827 Clean Tags were obtained after quality control,82,227 Clean Tags were obtained in primary culture(liquid medium)and 79,600 Clean Tags were obtained in subculture(solid medium);Finally,the pre-culture was mainly identified as Bacillus sp.,Staphylococcus sp.,Lactobacillus sp.,Paenibacillus sp.Sphingomonas sp.and the strains with higher abundance in subculture are Paenibacillus sp.,Staphylococcus sp.,Bacillus sp.,Turicibacter,Sphingomonas sp..(3)A total of 72.22 G of Raw data was produced by metagenome sequencing,and 54.53 G of Clean data was obtained after quality control.The results showed that Firmicutes,Actinobacteria and Proteobacteria were the main abundant species.Among them,Firmicutes with the most bacterial species were identified.The bacterial species mainly include:B.thuringiensis,B.cereus,Paenibacillus sp.and P.mucilaginosus.Secondly,the bacteria of Proteobacteria mainly include:Haliangium ochraceum and S.maltophilia;There are also Actinobacteria,mainly including R.erythropolis and Corynebacterium argentoratense.(4)Compare the results of culturomics with metagenome sequencing,it was found that the bacteria detected by both methods were B.cereus,Paenibacillus sp.,R.erythropolis.The bacteria appearing in both metagenomics and culturomics are the bacteria most likely to be infected in platelets.So B.cereus,Paenibacillus sp.and R.erythropolis,are most likely to be the contaminating bacteria of platelets.B.cereus,Paenibacillus sp.are generally skin flora,suggesting that the two bacteria may come from skin and that disinfection of skin is very important.On the other hand,the source of the detected Rhodococcus has not yet been determined.As to where it came from,further backtracking is required,but it is also required to strictly follow aseptic operation.In addition,more precautions should be taken against bacterial species detected by metagenomics.Conclusion:The process of microorganism culture,high-throughput sequencing and subsequent data processing has been established.The microorganism culturomics,the first generation sequencing of 16S rRNA gene,the 16S depth sequencing and the metagenome sequencing are used to verify the bacteria in platelets from different angles,and finally the bacteria in platelets can be accurately determined.It provides a new idea for highly sensitive detection of bacteria in platelets,and also makes a preliminary exploration for the development of a more automated culturomics detection system in the future.Through the comparative analysis of the results of microbial culturomics and metagenome,Proteobacteria and Actinobacteria detected but not cultured in high-throughput sequencing are bacteria that may exist in platelets.These information also provide a reference direction for improving the culturomics and thus detecting bacteria in platelets more widely.