香烟烟雾暴露对正常小鼠下呼吸道菌群分布的影响

Effect of cigarette smoke on bacteria distribution of lower respiratory tract in mice

目的探讨香烟烟雾暴露对正常小鼠下呼吸道菌群分布的影响。方法将10只雌性清洁级昆明系小鼠随机分成实验组和对照组各5只。实验组每天给予1次、每次2 h、每次14支香烟的烟雾暴露,持续70天。对照组正常饲养,不染毒。停止香烟烟雾暴露15天提取两组肺部细菌进行16S rRNA测序,检测其下呼吸道菌群组成并测算相对丰度。对下呼吸道菌群进行α多样性、稀释曲线、物种丰富度、PCA分析。结果对照组下呼吸道主要菌属按相对丰度从高到低依次为盐单胞菌(相对丰度70.8%)、Enterobacteriaceae_unclassified(相对丰度2.5%)、肠杆菌(相对丰度2.3%)、Pelagibacterium(相对丰度2.1%)、涅斯捷连科氏菌(相对丰度1.8%)、葡萄球菌(相对丰度1.4%)、芽孢杆菌(相对丰度1.2%)、Gemmatimonadaceae_uncultured(相对丰度1.1%)、副球菌(相对丰度0.8%)、红杆菌(相对丰度0.7%)、棒状杆菌(相对丰度0.6%)、Saccharibacteria_norank(相对丰度0.4%)、Zoogloea(相对丰度0.4%),实验组上述菌属相对丰度分别为46.8%、2.9%、2.5%、3.9%、0.9%、0.4%、0.3%、1.0%、19.3%、1.5%、1.5%、1.4%、1.2%。两组优势菌属十分相似。α多样性分析结果显示,两组Chao指数、Shannon指数、Simpson指数比较P均>0.05;两组Coverage指数为0.990~0.999。稀释曲线分析结果显示,随着样本序列的增大,细菌物种数变化不大。物种丰度差异性分析结果显示,两组丰度有差异且LDA值大于2的菌群共有10个。Metastats软件分析发现,在属的水平上,共16种菌属的相对丰度存在统计学差异;但存在统计学差异的菌群相对丰度均少于1%。说明香烟烟雾对优势菌属无影响,对少量非优势菌属存在影响。PCA分析结果显示,对照组菌群组成较为相似,观察组菌群组成则差异明显。结论香烟烟雾暴露对小鼠下呼吸道菌群的生物多样性无明显影响,但可影响部分非优势菌属的相对丰度。香烟烟雾暴露可能通过改变下呼吸道菌群组成引起宿主下呼吸道免疫反应改变,从而导致或者促进肺部疾病的发生。

Objective To determine the effect of cigarette smoke on the bacteria distribution of lower respiratory tract in mice. Methods Ten female clean mice were randomly divided into the control group and experimental group with 5 in each. The mice in the experimental group were given cigarette smoke exposure for continuous two hours one day for successive seventy days,with fourteen cigarettes burning out in the two hours. The mice in the control group were not exposed in cigarette smoke. Gene sequencing was performed by 16 S rRNA method after fifteen days without smoke exposure. The compositions of the lower respiratory tract bacteria and the bacteria relative abundances were measured. The α-diversity,dilution curve,species abundance,and PCA analysis were performed. Results The bacteria relative abundances of low respiratory tract in the control group were Halomonas（relative abundance 70. 8%）, Enterobacteriaceae-unclassified（2. 5%）,Enterobacterium（2. 3%）,Pelagibacterium（2. 1%）,Nesterenkonia（1. 8%）,Staphylococcus（1. 4%）,Bacillus（1. 2%）,Gemmatimonadaceae_uncultured（1. 1%）,Paracoccus（0. 8%）,Amaricoccus（0. 7%）,Corynebacterium（0. 6%）,Saccharibacteria_norank（0. 4%）,Zoogloea（0. 4%） arranged from high to low; while the bacteria relative abundances in the experimental group were 46. 8%,2. 9%,2. 5%,3. 9%,0. 9%,0. 4%,0. 3%,1. 0%,19. 3%,1. 5%,1. 5%,1. 4%,and 1. 2% corresponding with the control group. The advantage bacteria in the experimental group were similar to those of the control group. The α-diversity analysis showed that there was no statistic difference in the Chao index,Shannon index,and Simpson index（all P〈0. 05）. Coverage indexes of the two groups were at 0. 990-0. 999. Dilution curve suggested that bacteria species kept nearly stable with simple sequences increasing. Species abundance difference analysis suggested that ten species existed abundance differences and LDA 2 and sixteen species existed relative abundance difference by Metastats analysis. All the above species accounted for less 1%,which indicated that cigarette smoke could not influence the advantage bacteria but the disadvantage bacteria. PCA showed that the compositions of bacteria in the control group were similar,but the experimental group varied significantly. Conclusions Cigarette smoke have little influence on the biodiversity of lower respiratory tract bacteria but can change the relative abundance of disadvantage bacteria. Cigarette smoke may change immunoreaction of lower respiratory tract by affecting the bacteria,further cause or promote the development of lung diseases.