Study Of Microbial Community Of The Lower Respiratiory Tract Of Patients With Idiopathic Interstitial Pneumonias(IIPs) Based On Metagenomic Data

PARTⅠObjective:This study aimed to find a better method to enable a higher concentration of microbial DNA to be extracted from the BALF samples.Methods:(1) Two individuals with IIPs were selected. BALF sample of each individual was divided into 2 equal quantities, one for group frozen, one for group fresh. Samples in group fresh were immediately performed DNA extraction after being collected; Samples in group frozen were stored in -80℃ through being pretreated and would be performed DNA extraction.(2) We selected twelve individuals with IIPs, BALF samples and mouthwash samples of all subjects were collected. All subjects were randomly divided into 3 groups according to different DNA extraction methods:group kitl, group kit2, and group kit3. BALF samples were marked as A and mouthwash samples as B. DNA extractions were performed according to manufacturer’s instructions.(3) DNA libraries were constructed according to the modified manufacturer’s instructions (Illumina). Using Hiseq 2500 to perform metagenomic sequencing. Raw data was uploaded to MG-RAST and analyzed.Results:(1) The ratio of human genome in clean reads of each DNA sample from frozen BALF samples was remarkably smaller than those from fresh BALF samples (Patient 1:59.80% vs.68.60%, Patient 2:47.91% vs.66.89%, respectively). For each individual, numbers of hits from frozen samples by same database are more than those from fresh samples. There was very tiny difference on the top 50 most abundant classified phyla between fresh BALF sample and frozen BALF sample from the same individual. There was no significant difference on the principal bacterial community structure between fresh BALF samples and frozen BALF samples by comparing the bacterial community structure of fresh BALF samples with frozen BALF samples.(2) DNA concentrations of DNA samples isolated with kitl with Benzonase were significantly lower than those isolated with the other two kits for BALF and mouthwash samples. The ratio of human genome in clean reads of samples isolated with kitl with Benzonase was remarkably smaller than those isolated with kit2 and kit3. The richness of bacteria in group kitl A was significantly higher than group kit2A and kit3A (richness: P=0.009).Conclusions:Cryopreservation of BALF samples can enable a higher yield of microbial DNA from samples with a higher fraction of host cells to be obtained, and will not affect the principal bacterial community structure. A microbial DNA extraction method with pretreatment of depletion of host nucleic acid by Benzonase can reduce ratio of host DNA in total DNA to be obtained, and will not affect the principal bacterial community structure.PART ⅡObjective:This report aimed to identity and compare the characteristics of the microbial community in lower airways of IIPs with healthy lung.Methods:We collected bronchoalveolar lavage fluid specimens from 38 healthy controls (group control including 15 smokers and 23 non-smokers), and 17 IIP patients (group case). Using QIAamp DNA Microbiome Kit to perform DNA extractions. DNA libraries were constructed according to the modified manufacturer’s instructions (Illumina). Using Hiseq 2500 to perform metagenomic sequencing. Raw data was uploaded to MG-RAST and analyzed.Results:Proteobacteria dominated the lower airways microbial communities of IIPs and healthy lung, followed by Firmicutes, Actinobacteria and Bacteroidetes at the phylum level. At the genus level, Caulobacter, Haemophilus and Bacteroides were significantly decreased in group case compared with group control, and Achromobacter, Bordetella and Rhodococcus were significantly increased in IIPs. There were 6 species increased significantl (Achromobacter xylosoxidans, Methylobacterium extorquens, Achromobacter piechaudii, Bordetella bronchiseptica, Bordetella parapertussis, and Rhodococcus erythropolis), and 7 species decreased significantly (Actinomyces odontolyticus, Phenylobacterium zucineum, Neisseria meningitidis, Caulobacter vibrioides, Caulobacter sp. K31, Haemophilus influenza and Caulobacter sp.) in IIPs. The relative abundance of Propionibacterium was significantly negatively correlated with the concentration of C reactive protein in blood, Pseudomonas and Burkholderia were significantly negatively correlated with erythrocyte sedimentation rate and FEV1/FVC%, but significantly positively correlated with FVC%, FEV1%, VC% and FEV1; Bradyrhizobium was significantly negatively correlated with FEV1/FVC%; Prevotella was significantly positively correlated with FEV1/FVC%, and was significantly negatively correlated with FEV1.Conclusion:Patients with IIPs might share the similar dominant phyla to the healthy controls, but there could be dysbacteriosis in the lower respiratory tract of patients with IIPs at the genus and species level, and some of genera are significantly correlated with pulmonary function and inflammatory factors.