High Throughput Analysis For Pathogens At The Nucleic Acid Level In Neonatal Penumonia

Objective The purpose of this study is to apply two high throughput analysis for pathogens at the nucleic acid level in neonatal penumonia: Easy Operating Pathogen Microarray(EOPM)technology and 16 S rRNA gene high throughput sequencing technology.Materials and Methods1.Study Population: The newborns who suffered from pneumonia were recruited from the Department of Neonatology of the Guangdong Women and Children's Hospital,Guangzhou Medical University between December 2015 and April 2016.Tracheal aspirates were collected from the patients who were mechanically ventilated because of pneumonia.The protocol for tracheal aspirates collection consisted of instillation of 0.5-1 ml of sterile isotonic saline into the infant's endotracheal tube,and suctioning of the fluid into a sterile mucus trap.Samples were stored frozen in-80 ? until DNA extraction.Clinical data and pathogenic examination were also collected for those who were in in this study.2.The procedure of Easy Operating Pathogen Microarray: Sample were prepared and microbial nucleic acids were extracted from tracheal aspirates and to be used for EOPM analysis.The arrays were scanned using a dual-laser scanner and the images were extracted and analyzed using Feature Extraction software.Finally,we need to input raw microarray data into the EOPM software for the statistical enrichment analysis of pathogens.The pathogen positive samples were verified by PCR and DNA sequencing.3.The procedure of 16 S rRNA gene high throughput sequencing: The terminal sequences of the bacterial rRNA gene V3-V4 region are conserved among different bacteria.Sequence libraries were constructed by amplification of the V3-V4 regions of the bacterial 16 S rRNA gene,according to the Illumina manufacturer's instructions.Bioinformatic analysis of the sequences data: The sequences were clustered into operational taxonomic units(OTUs);Alpha diversity analysis.All the data were taxonomically grouped using the Ribosomal Database Project(RDP)classifier and Greengenes database.Results1.Neonatal demographic and clinical characteristics: A total of 24 tracheal aspirates were obtained from 22 newborns during the study period.Those of them were suffered from pneumoina.Examination of pathogen positive were found in 13 out of 24(54.2%).11 out of them were bacterial cultures positive,and 2 were respiratory syncytial virus(RSV)positive by using PCR.2.The outcome of Easy Operating Pathogen Microarray: RSV was found to be significantly enriched in four samples.Their highest ratios of cy5/cy3 were74.4,60.8,72.4 and 11.5,respectively.And their enrichment p-values were all < 0.01.Bacterial positive were found in four samples.two of them were Streptococcus,the highest ratios of cy5/cy3 were 31.6,9.1,respectively,enrichment p-values were all <0.01.Bacillus was found in one sample,the highest ratio of cy5/cy3 was 39.9,enrichment p-value was < 0.01.Two samples was found to be pathogens positive,one was bacteria while another was fungi.But both of them were not assigned to the genus or species level.We verified RSV in four samples by PCR targeted to a region of RSV genomic sequence and DNA sequencing.3.Operational Taxonomy Units(OTUs)clustering program and alpha diversity analysis: The average number of OTUs in 24 samples were 797(range:47-2524).The average sequences number were 10624(range: 144-34392)per sample.The average Shannon index was 4.0(0.99-7.58)and Simpson index was0.71(0.19-0.99).Rarefaction curves were used to estimate whether the number of sequences is sufficient to cover all species and estimate species richness.Rarefaction curves showed that the saturated shapes of the rarefaction curves indicated that sequencing depth has covered all species in the sample.4.The total classification of bacteria: There are 252366 sequences produced and 16220 OTUs in 24 samples.251859(99.8%)could be classified into the bacterial domain,out of which 176087(69.9%)sequences could be assigned to the genus level including 209 bacterial genera.The classification level of each OTU ratio was phylum,class,order,family and genus at 89.8%(14559/16220),89.8%(14558/16220),89.7 %(14550/16220),83.9 %(13603/16220)and 62.3 %(10097/16220),respectively.The sequences number of genus Streptococcus accounted for 25% of the total sequences,while genus Rothia sequeces number accounted for 8%.The genus Bacillus,Acinetobacter,Klebsiella and Pseudomonas account for 6%,6%,5%,5%,respectively.5.The pathogen profile at phylum and genus level was analyzed: There were 14 out of 24 samples had a predominance of Proteobacteria,while 7 had a predominance of Firmicutes.Proteobacteria and Firmicutes were distributed in all of the samples.Bacteroidetes occurred in 21 samples and Actinobacteria occurred in 18 samples.At genus level,there were 8 dominant genera in 11 samples: Pseudomonas,Klebsiella,Streptococcus,Acinetobacter,Haemophilus,Ureaplasma,Rothia and Bacillus.The frequency of the sequeces of genus of bacterial detected in 24 samples were,in order,Streptococcus 87.5%(21/24),Pseudomonas 87.5%(21/24),Acinetobacter 83.3%(20/24),Methylobacterium 70.8%(17/24),Paenibacillus70.8%(17/24)and Staphylococcus 66.7%(16/24).6.The genus Streptococcus and genus Bacillus were identified by PCR:The sequences of genus Streptococcus were founded in 21 samples.There are 7samples which the sequences of gunus Streptococcus accounted for more than 10%in each sample.we performed Streptococcus sp.,Streptococcus agalactiae,Streptococcus pneumoniae and Streptococcus mitis specific PCR targeted to a conserved region of each bacterial genomic sequence.The results show that Streptococcus sp.specific PCR were tested positive in 6 samples.The cfb gene of Streptococcus agalactiae specific PCR were tested positive in 2 samples.We performed nine genes and four toxin genes(p X01-110(pag A),p X01-122(cya),p X02-56(cap A),p BC218-0129)of Bacillus cereus specific PCR.And we found that nhe A,nhe B,nhe C,p BC218-0129 were tested positive,respectively.All the positive PCR products were further verified by DNA sequencing.Conclusion1.This study reveals the complicated composition of pathogens in the tracheal aspirates of neonatal pneumonia.The dominant pathogens include Streptococcus,Pseudomonas,Acinetobacter,etc.In addition to considering the bacterial pathogens infection,we should not ignore the virus infection.It is highly important to detect virus in those newborns with pneumonia,especially the RSV.2.EOPM can detect respiratory viruses effectively.But the detection ability of bacteria remains to be improved and optimized in the future.EOPM should be tested for its sensitivity and specificity.This mean each probe should be confirmed with specific positive and negative samples.3.Compared to the traditional bacterial culture method,16 S rRNA gene sequencing not only can detect the traditional bacterial pathogens which are identification from culture methods,but also can detect those pathogens which could not acquired by culture.16 S rRNA gene sequencing technology can provide us with an overview of the complex microbial communities in a patient with pneumonia.This study shows that 16 S rRNA gene sequencing technology is an effective method to analyze the complex microbial communities of those newborns with pneumonia.And it can avoid the bias that the routine methods can only identify limited and typical pathogens.4.When the traditional methods can not detect those pathogens and the clinical treatment is poor,high-throughout molecular biology methods should be considered to be used to screen pathogens,which can help the clinical treatment.