| Background：Asthma is a chronic inflammatory disease of the airways, the potential link betweenmicrobial infections and asthma is now thought to be environment, immunity and geneticfactors. Some epidemiological studies have found that bacteria have now been recognizedas a possible cause of asthma, whereas bacteria microbes have been identified inexacerbation cases. The potential role of bacterial colonization or infection of thebronchial mucosa in the pathogenesis of asthma has been raised by several recent reports.Asthmatic patients were noted to have more proteobacteria in their respiratory system,whereas nonasthmatic individuals were seen to have more bacteroidetes. Although therole of microbial infection in asthma pathogenesis remains unclear, studies to date haveprimarily applied culture-based or targeted molecular approaches to identify specificbacterial species of interest in the airways. Recent developments in defining the humanmicrobiome have demonstrated that the composition of bacterial communities colonizingmucosal surfaces, rather than simply the presence of individual species, can be importantin defining states of health or disease. Culture-independent microbiota profiling based onsequence polymorphisms in the16S ribosomal RNA (16S rRNA) gene, which is presentin all bacteria, has been widely applied in environmental ecologic studies and in studiesof the human”superorganism”.16S rRNA-based phylogenetic analysis throughsequencing or phylogenetic arrays permits detection of uncultured or fastidious bacteria,providing information on community composition without a priori knowledge of thosepresent.Currently, the asthma-associated differences in microbial composition of induced sputumby using primers specific for the V3-V5hypervariable region of bacterial16s rRNA has been raised by several abroad reports, but there are no related reports in china. In thisstudy, for the first time, we established the methodology of how to obtain induced sputumand used the high-throughput454sequencing technology to obtain estimates of bacterialdiversity in induced sputum from asthmatic and healthy people.ObjectiveWe sought to characterize and compare the microbiome of induced sputum in asthmaticand healthy subjects.MethodsThirty-nine asthmatic and eighteen healthy controls were studied. All participantsunderwent clinical assessment, skin prick testing, spirometry and sputum induction.Before sputum induction, asthmatic patients requested to evaluate their disease statuswith asthma control test(ACT). The sputum was used for bacterial DNA extraction andidentification. Total DNA was amplified by using primers specific for the V3-V5hypervariable region of bacterial16S rRNA. Samples were barcoded, and sequenced withthe454GS FLX sequencer. Sequences were assigned to bacterial taxa by comparingthem with16s rRNA sequences in the Ribosomal Database Project.Results1.Basic information of the study subjects①Thirty-nine asthmatics and18healthy subjects were enrolled in the study. Sputumwas successfully induced in27asthmatics (69.2%) and9healthy controls (50%).②Asthmatics with an ACT score of22.8±1.8had lower%FEV1predicted(71.3±21.0%vs99.4±6.0%, p<0.001), FEV1/FVC%（65.7±10.4%vs82.9±3.4%,p＜0.01）and higher sputum eosinophils (33.6±25.9%vs0.8±0.6%, p<0.001),average age（48.0±10.9vs37.4±4.8, p＜0.01）.2.Quality of sputumThere was no difference in sputum weight（0.14±0.031g vs0.13±0.025g, p=0.474）, cell viability（88.4±4.6%vs89.0±2.6%, p=0.892） and Squamous cel（l2.0±2.1%vs2.0±1.2%, p=0.541）between groups of asthmatic and healthy people.3.Diversity Analysis of SpeciesShannon index were decreased in samples from asthmatic[3.9(3.6-4.4) vs4.2(3.8-4.5),p=0.267], although this was not statistically significant. OTU production werefound more frequently in samples from healthy people （92.3±31.5vs107.9±15.2,p=0.016）.4.Bacterial spectrum analysis(Phylum)①Bacterial compositionAll sputum samples contained6major bacterial phyla: Firmicutes, Proteobacteria,Bacteroidetes, Actinobacteria, Fusobacterium and Spirochaetes, with the first3phylaaccounting for more than90%of the total sequences.②Asthma group vs Healthy controls groupThere was no difference between groups of asthmatic and healthy people: Bacteroidetes(27.0%±13.1%vs33.1%±10.4%, p=0.223), Firmicutes (33.8%±12.7%vs29.0%±13.8%, p=0.349),Proteobacteria [26.1%(13.1%-33.9%) vs23.8%(12.3%-47.9%), p=0.818], Fusobacteria [3.2%(1.7%-8.1%) vs5.6%(4.9%-7.7%),p=0.075] and Spirochaetes [0.2%(0.1%-0.9%) vs0.4%(0.2%-1.2%), p=0.216]③Severe asthma group vs Mild asthma groupThere was no difference between groups of severe asthma and mild asthma:Bacteroidetes (23.3%±12.8%vs30.4%±13.0%, p=0.202), Firmicutes (32.7%±14.4%vs34.9%±11.4%, p=0.678), Proteobacteria (30.5%±22.8%vs24.3%±14.9%, p=0.442),Fusobacteria [2.9%(1.7%-8.1%) vs3.4%(1.9%-8.0%), p=0.712], Actinobacteria [2.4%(1.5%-5.8%) vs2.0%(1.2%-4.4%), p=0.460] and Spirochaetes [0.2%(0.0%-0.3%) vs0.3%(0.1%-1.1%), p=0.147].④The Actinobacteria were significantly higher in asthma group than that in healthycontrol group [2.2%(1.4%-4.7%) vs0.6%(0.4%-1.9%), P=0.02].5.Bacterial spectrum analysis(Family)The Cellulomonadaceae(2.1783±2.0111vs0,p=0.014), Actinomycetaceae(1.6593±0.2468 vs0.8170±0.282,p=0.046), Methylobacteriaceae(0.0876±0.0359vs0.0151±0.0093,p=0.041), Phyllobacteriaceae(0.0682±0.0343vs0.0010±0.001,p=0.032), Brucellaceae(0.0278±0.0156vs0.0014±0.0014,p=0.027), Bradyrhizobiaceae(0.0155±0.0132vs0,p=0.047)and Burkholderiaceae(0.0062±0.0038vs0,p=0.047)were significantly higherin asthmatic than that in healthy control group.Conclusions1. OTU production were decreased in samples from asthmatic, especially in severeasthma group. Shannon index were decreased in samples from asthmatic, althoughthis was not statistically significant.2. All sputum samples contained6major bacterial phyla(Firmicutes, Proteobacteria,Actinobacteria, Fusobacterium, Spirochaetes and Bacteroidetes): Firmicutes,Proteobacteria and Bacteroidetes accounting for more than90%of the totalsequences. Actinobacteria were present in higher proportions in asthmatic patients,especially in Severe asthma group. In contrast, Bacteroides were found morefrequently in samples from healthy control group, although this was not statisticallysignificant.3. Characteristics of the severe asthma group had declining trend in Shannon index,higher Actinobacteria, lower OTU production.Compared with the severe asthmagroup, Bacteroides were found more frequently in samples from healthy controlgroup and non-severe asthma group, although this was not statistically significant.4. The Cellulomonadaceae, Actinomycetaceae, Methylobacteriaceae, Phyllobacteria-ceae, Brucellaceae, Bradyrhizobiaceae and Burkholderiaceae were significantlyhigher in asthmatic than that in healthy people.5. The corresponding biological effect on airway inflammation remains to bedetermined. |
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