Identification And Analysis Of Bacteria Caused Bacterial Meningitis In Cerebrospinal Fluid Based On Next-Generation Sequencing Technology

Objective:At present,the methods of bacteria diagnosis based on bacterial culture and traditional molecular techniques for bacterial meningitis(BM)are low in flux,low in sensitivity,low in positive rate,and time-consuming,affecting the choice of the best treatment plan for patients.And recent research found that[1],There were multiple pathogens coexisting n the cerebrospinal fluid of patients with BM.Therefore,the purpose of this study was to use a new etiological diagnosis method that it didn’t depend on bacterial culture.The Next-Generation Sequencing(NGS)technology was used to screen pathogens that cause BM,and to analyze bacterial population abundance,bacterial composition and microbial diversity,and to provide a theoretical basis for the diagnosis of bacterial infection in clinical BM patients.Simultaneously,we optimized a set of methods that can effectively trace amounts of DNA in samples with a low volume and only a trace amount of pathogenic microorganisms.so that trace DNA can meet the requirements of routine sequencing,providing more data for later bioinformatics analysis and obtaining as much pathogen-related information as possible.Methods:Firstly,we established a method for the extraction of trace bacterial DNA using the"simulated cerebrospinal fluid" samples in this study.The establishment of trace DNA extraction methods was mainly through the fol lowing tests:the selection of two kits that is the QIAamp(?)DNA Mini Kit and the QIAamp(?)DNA Microbiome kit,both of which were suitable for extracting bacterial DNA from CSF.And the exploration of the bacterial lysis time.And analyzing the effect of ethanol nucleic acid precipitation vector on bacterial DNA extraction.Secondly,the methodological validation was performed in clinically cultured positive CFS patients using a previously established method.Finally,31 CSF patients with clinically diagnosed BM were selected for DNA extraction and gene library construction according to our established method of DNA extraction,and DNA library was sequenced by using 16SrDNA Metagenomic sequencing technology on Ion Torrent PGM platform.Pathogen species,bacterial abundance,flora composition and species diversity of infected CSF were analyzed based on sequencing results.Results:The method of DNA extraction and high-throughput sequencing identification of pathogenic bacteria was validated.The results showed that the pathogen identified by 16S rDNA sequencing was basically the same as that identified by clinical bacterial culture,indicating that the established trace DNA extraction method has practicality.It also has illustrated the feasibility of high-throughput sequencing in the detection of BM pathogens.To identify the pathogens of infection in 31 BM patients:A total of 55 bacterial flora were identified at the phylum level and a total of 596 bacterial species at the genus level were identified.Among them,Bacteria mainly include Firmicutes,Proteobacteria,Actinobacteria,Bacteroidetes and Fusobacteria at the phylum.Predominant species were Streptococcus,Escherichia,Pseudomonas,Peptostreptococcus,Acinetobacter,Prevotella,Neisseria and Haemophilus influenzae at the genus.It showed that pathogenic bacteria was not composed of only a single bacteria or several bacterial,the incidence of infection may be the result of the interaction of multiple bacteria.The Alpha diversity Shannon Index and Simpson Index were approximately 6.80 and 0.95 respectively,indicating that the species diversity was low and the complexity of bacterial species was not high in these CSF samples.Analysis of Beta diversity-related indicators revealed that there were significant regional differences in the types of pathogens,species abundance and species composition in different BM patientsConclusions:In this study,a set of effective methods for DNA extraction and sequencing in trace amounts of CSF can accurately detect pathogenic bacteria in patients with clinical BM.It provided a theoretical basis for the change of infection spectrum and the diagnosis of pathogenic bacteria in patients with clinical BM.The study found that it showed that pathogenic bacteria was not composed of only a single bacteria or several bacterial,the incidence of infection may be the result of the interaction of multiple bacteria,And the bacterial infection profile in patients with bacterial meningitis has regional differences.