Study On The Correlation Between Air Particulate Matter And Airway Bacterial Community Structure In COPD Patients

Objective:To analyze the evolution of bacterial community structure in sputum of AECOPD patients by 16S r RNA gene sequencing,and to preliminarily explore the correlation between bacterial community structure in atmospheric particulate matter and respiratory tract bacterial community structure of AECOPD patients,so as to provide basis for clinical introduction of respiratory tract bacterial community as a new target.Methods:1.Clinical subjects:A total of 15 AECOPD patients hospitalized in respiratory and critical care Medicine Department of Baogang Hospital of Inner Mongolia from December 2020 to February 2021 were randomly selected and divided into pre-treatment group（d1 group）and treatment day 3 group（d3 group）.Fresh sputum samples were collected by spontaneous sputum or induced sputum on the first day and the third day of treatment respectively.2.Experimental subjects of atmospheric particulate matter:They were divided into coarse particulate matter group(PM₁₀)and fine particulate matter group(PM_2.5).Two sampling sites were set up in Baotou,Inner Mongolia,with a sampling height of about 10m from the ground.The sampling time was the same as that of clinical case collection,and each sampling time lasted for 23h（10:00 am-9:00 am of the next day）.An integrated atmospheric sampler（KB-6120）was used to collect 8 samples of coarse particulate matter（PM10）and fine particulate matter（PM2.5）.3.Experimental content:（1）Clinical indicators of lung function,WBC,NEU,CRP,PCT and other clinical indicators were collected from the selected patients,and a comparative study was conducted before and after the observation indicators.（2）Genomic DNA extraction,PCR amplification,database construction and sequencing were performed on all sputum samples and atmospheric particulate matter samples.After completing the quality control process,the microbial community structure was analyzed and described.（3）Species diversity and abundance in d1 and d3 groups were evaluated byαdiversity（Wilcox rank-sum test）.βdiversity（obtained by NMDS analysis）was analyzed to investigate the difference in species complexity between d1 and d3bacterial communities.Linear discriminant analysis effect size（LEFSE）was used to obtain the species difference between d1 and d3 groups.（4）Spearman correlation analysis was used to evaluate the relationship between dominant bacteria and clinical indicators in d1 group,and the correlation between PM_2.5bacterial community and the bacterial community in d1 group sputum samples.Pheatmap function in pheatmap software package was used for visualization.（5）PICRUST analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes（KEGG）database to assess microbiome function in PM_2.5and PM₁₀samples.Results:1.Clinical experiment:（1）WBC,NEU and CRP of AECOPD patients in d3 group were significantly lower than those in d1 group（T=3.47,2.68,3.32,all P<0.05）;PCT in d3 group was lower than that in d1 group（t=1.87,P>0.05）,with no statistical significance.（2）Sputum samples of patients were detected by 16S r RNA gene sequencing method,and it was found that,at the phylum level,the dominant phylum of patients in d1 group and d3 group were:Firmicutes（42.71%and 50.67%）,Proteobacteria（35.62%and 23.84%）,Bacteroidetes（13.32%and 12.98%）,Actinobacteria（7.57%and 9.83%）and Fusobacteria（0.59%and 1.15%）,etc.The relative abundance of Proteobacteria in d3 group was significantly lower than that in d1 group（t=2.83,P<0.05）.（3）At the genus level,the dominant bacteria in d1 and d3groups were Streptococcus（27.87%and 25.88%）,porphyromonas（7.44%and 3.38%）,Neisseria（6.72%and 3.41%）,Rothia（6.70%and 3.65%）,prevotella（6.29%and13.64%）,etc.Among all bacteria,the relative abundances of Rothia,Neisseria and Haemophilus in d3 group were significantly lower than those in d1 group（t=2.55,2.36 and 2.95,all P<0.05）.The relative abundance of Prevotella in d3 group was significantly higher than that in d1 group（t=-2.29,P<0.05）.（4）There was a significant positive correlation between FEV1/FVC and the relative abundance of Veillonella in d1 group.The relative abundance of Staphylococcus was positively correlated with CRP（r=0.85,0.59,P<0.01）.（5）Shannon index and Simpson index in d1 group and d3group were 2.14±0.62 and 2.24±0.61,0.79±0.15 and 0.75±0.17,respectively.Chao1and ACE index of d1 group and d3 group are 80.37±33.74 and 74.32±23.71 and78.97±27.74 and 76.51±19.74,respectively.（6）The distance between d1 group and d3group was less than 0.2 Stress.（7）Acinetobacter and Bacillus were the most abundant sputum flora in group d1,while Escherichia,Nitrospirae,Tannerella and Pseudonymella were the most abundant sputum flora in group d3.2.Atmospheric particulate matter experiment:（1）The dominant bacteria in PM_2.5and PM₁₀were Proteobacteria（50.98%and 54.64%）,Bacteroidetes（35.69%and 34.61%）,Actinomycetes（5.51%and 5.46%）and Firmicutes（5.42%and 3.26%）,respectively.（2）The dominant bacterial genera in PM_2.5and PM₁₀were Sediminibacterium（24.75%and 28.84%）,Bradyrhizobium（20.96%and 19.17%）,Methylovirgula（9.85%and 7.34%）,Rhodanobacter（3.30%and 3.74%）and Wolbachia（3.18%and7.27%）,etc.（3）At the level of phylum,there were 17 species of dominant bacteria overlapping in the sputum samples of patients in PM_2.5and d1 groups,and the dominant bacteria in relative abundance from large to small were Proteobacteria（40.96%）,Firmicutes（29.74%）,Bacteroides（21.10%）,Actinobacteria（6.86%）,etc.At the genus level,there were 118 species of bacteria overlapping sputum samples of patients in PM_2.5group and d1 group,and the dominant bacteria were Streptococcus（18.28%）,Sediminibacterium（11.19%）,Bradyrhizobium（8.63%）,Porphyromonas（5.01%）,Prevotella（4.42%）,etc.3.Spearman correlation analysis was conducted between the main bacteria of PM_2.5and the sputum specimens of d1 group that Sediminibacterium of PM_2.5and sputum specimens in d1 group of Porphyromonas,Neisseria,Staphylococcus was significantly positively related,and Streptococcus,Prevotella bacteria were significantly negative correlation（both|r|>0.5,p<0.05）;PM_2.5slow of Bradyrhizobium in the genus group with d1 sputum specimens of Porphyromonas,Haemophilus was significantly positively related,and Streptococcus has significant negative correlation（both|r|>0.5,p<0.05）;PM_2.5in the sputum specimens of the genus Mycobacterium and d1 group Neisseria bacterium,Haemophilus was significantly positively related,in walter bacteria has significant negative correlation（both|r|>0.5,p<0.05）.4.In the prediction of PM_2.5and PM₁₀functions,the second-order KEGG pathway was found to be dominated by replication and repair,translation,nucleotide metabolism,glycan biosynthesis and metabolism,ABC transporter and secretion system,and other secondary metabolites biosynthesis.Conclusions:1.Clinical studies showed that:（1）The bacterial community structure of sputum microbiota in AECOPD patients was similar before and after treatment,and the relative proportion of dominant phylum and dominant genus was different.（2）The diversity of sputum microbiota before and after treatment was not obvious.（3）After treatment,the relative abundance of Proteobacteria and Rothia,Neisseria,Haemophilus,Acinetobacter and Bacillus decreased significantly;The relative abundance of Prevotella increased significantly.（4）There was a significant positive correlation between the relative abundance of Veillonella and FEV1/FVC,Staphylococcus and CRP in d1 group.2.Experimental results of atmospheric particulate matter showed that:（1）PM_2.5and PM₁₀had similar bacterial community structure,and the relative proportion of dominant phylum and dominant genus was different.（2）In the functional prediction,replication and repair,translation,nucleotide metabolism,glycan biosynthesis and metabolism,ABC transporter and secretion system,and other secondary metabolites are the main biosynthesis.3.There was a significant correlation between the dominant bacteria in PM_2.5and the dominant bacteria in the sputum samples of patients in d1 group.