Changes In The Pulmonary Microbiota And Its Metabolites In Children With Precocious Heart Disease With Pulmonary Hypertension

Objective(s):To investigate the diversity of pulmonary microbiota in children with Pulmonary arterial hypertension associated with the congenital Left-to-Right shunt(PAH-LTRS)and its microbial metabolites on PAH-LTRS.The search for lung microorganisms and metabolites that can be used as potentially effective biomarkers of PAH-LTRS disease can provide some reference for the diagnosis as well as the treatment of PAH-LTRS.Methods:In this study,the 68 age-matched subjects included were divided into three different sample groups(PAH-LTRS patients group,LTRS patients but no PAH group,and REF group)according to inclusion as well as exclusion criteria.Of these 68 samples,some BALF samples were not sufficient for simultaneous microbial diversity analysis as well as metabolomics analysis.Therefore 47 were dispersed in different groups for microbial diversity analysis and 59 for metabolomic analysis.1.Bronchoalveolar lavage fluid(BALF)was collected from 68 subjects according to an established standard protocol.2.Total bacterial genomic DNA was extracted from the BALF samples and tested for DNA concentration and purity.3.PCR amplification,identification,purification and characterization of PCR products were performed on the extracted bacterial total genomic DNA,and then a genomic library was constructed.4.The constructed genomic library is analyzed by 16 S r RNA high-throughput sequencing.5.The sequencing data are analyzed for microbial diversity,i.e.species composition analysis,Alpha diversity analysis and Beta diversity analysis,and then the high-throughput sequencing data are analyzed for species differences using LDA Effeft Size analysis to find the dominant or specific groups of bacteria.6.Finally,metabolomics was used to explore the metabolomic alterations in the lung microbiota of PAH-LTRS and to investigate the correlation between the lung microbiota and metabolites in order to find diagnostic markers that can be used as a diagnostic marker for PAH-LTRS.Results:We found no significant changes in microbial community Alpha diversity analysis in PAH-LTRS.Microbial composition analysis,Beta diversity analysis and PLS-DA analysis showed that Lactobacillus and Alicycliphilus were the dominant genera in PAH-LTRS.Metabolomic analysis showed significant metabolic alterations in the PAH-LTRS sample group.Correlation analysis of microorganisms and their metabolites suggested that 2-piperidone,Moraxella,Lactobacillus and Porphyromonas are expected to be diagnostic markers for PAH-LTRS in children.Conclusion(S):Alpha diversity of lung microorganisms was similar in patients with PAH-LTRS.Compositional and Beta diversity analyses showed that the relative abundance of microbiota was different between groups.significant metabolic alterations occurred in the PAH-LTRS sample group.2-piperidone,Moraxella,Lactobacillus and Porphyromonas are expected to be diagnostic markers for PAH-LTRS in children.