| Objectives:1.The species composition,interspecies relationships,community diversity and function prediction of the middle ear microbial community in chronic suppurative otitis media(active stage)and middle ear cholestatoma were obtained by 16SrRNA sequencing method.2.The species composition,interspecies relationships,community diversity,and function prediction of the middle ear microbiota in the middle ear mucosal tissue specimens of chronic otitis media at quiescence and fresh tympanic membrane perforation in the control group(relative "non-inflammatory state")were obtained by 16SRRNA sequencing.3.Bioinformatics and statistical methods were used to compare the results of microbial community composition,species composition,species diversity abundance and microbial function of the middle ear in the inflammatory state of chronic otitis media and the relative "non-inflammatory state" analyzed by the above 16SRRNA sequencing method.To explore and identify pathogenic microbial communities and species composition in the inflammatory process of chronic otitis media,as well as the pathogenic bacterial colonies.Methods:In this study,granulation specimens of middle ear from patients with active chronic suppurative otitis media and cholesteatoma specimens from patients with middle ear cholesteatoma who underwent surgery in the First Affiliated Hospital of Kunming Medical University from June 2019 to December 2020 were collected,and the above specimens were confirmed by postoperative pathological diagnosis results simultaneously.At the same time,tympanic mucosa specimens(static phase of chronic purulent otitis media/traumatic perforation of tympanic membrane)were collected as a control group in a"relatively non-inflammatory state".A total of 26 specimens were collected,including 6 in group T1(" relatively non-inflammatory state"control group),13 in group T2(middle ear cholestatoma specimen group),and 7 in group T3(middle ear granulation group with chronic suppurative otitis media(active stage).Specific primers in 16S V3-V4 region were designed to amplify the specific region,and a 420bp amplified fragment was obtained.The two ends of 2X250bp were sequenced using Novaseq platform,and a longer sequence was obtained by splicing.Finally,the biological information of 16SrRNA sequencing results was analyzed.Results:1.Clustering OTU and species annotation:A total of 13,942 OTU were generated from 26 samples.Proteobacteria,Bacteroidetes and Firmicutes were the main bacteria groups in the "relatively non-inflammatory state"T1 group at the level of Phylum classification,which involved 7 bacterial genera.They were Lysobacter,Flavobacterium,Arenimonas,and 10 subgroups,respectively 10)Luteimonas,Altererythrobacter and Staphyiococcus,Proteobacteria,Bacteroidetes and Acidobacteria were the main bacteria groups of middle ear cholestatoma T2 group and T3 group of chronic suppurative otitis media(active stage),which involved 6 bacteria genera.They were Lysobacter,Flavobacterium,Arenimonas,and 10 subgroups,respectively 10),Luteimonas and Altererythrobacter belong to the three genera.The abundance of Staphyiococcus in T1 group was higher than that in T2 and T3 group.2.Uniunique bacterial flora analysis of the samples:there were 9228 OTU species in Ti,12033 OTU species in T2,and 9991 OTU species in T3,and a total of 7187 OTU species were shared by T1,T2,and T3.626 OTU species were unique to Ti,2233 OTU species were unique to T2,and 926 OTU species were unique to T3.3.Comparison of sample flora diversity:T3 group had the highest Simpson index,T2 group had the lowest Simpson index(P=0.019),and there was no statistical difference in Chao index between T1 group,T2 group and T3 group(P=0.24).The T3 group had the smallest diversity of middle ear colonies,while the T2 group had the largest diversity.4.Analysis and comparison of sample flora:at the phylum level,Deinococcus-thermus,Elusimicrobia and WS1 were the main colonies that differed between the T1 and T2 groups.At the phylum level,the main difference colonies between T1 and T3 groups were Gemmatimonadetes,Elusimicrobia,Fibrobacteres and Hydrogenedentes.5.LEFSE analysis results:in T1 group,the abundance of the following microflora in tympanum of patients in the "relatively non-inflammatory state" control group was significantly increased and had clinical significance:Bacilli→Bacilli(Bacillales),Staphylococcaceae 一 Staphylococcus → Staphylococcusaureus,WS,Cloacimonadaceae,Thal Assospira);In the T2 group,the abundance of several microflora in the middle ear of patients with cholesteatoma type of chronic suppurative otitis media was significantly increased and had clinical significance:FamilyXiFamily and Genus W5053;In group T3,the abundance of several bacterial communities in the middle ear of patients with chronic suppurative otitis media(active stage)was significantly increased and had clinical significance:Planctomycetes→Planctomycetacia and Betaproteobacteriales.6.Prediction of sample colony function:there were 6 primary pathway functions in the samples,including cellular process,environmental information process,gene information process,human disease,metabolomics,and organic system,and 42 secondary pathway functions in the samples.Samples from the middle ear cholesteatoma group,in immune system,folding and classification and degradation,glycan biosynthesis and metabolism,digestive system,metabolism of terpenoids and polyketones,environmental adaptation,nucleotide metabolism,replication and repair,translation,infectious diseases:Bacterial,transcriptional,endocrine and excretory functions were highly expressed,while "relatively non-inflammatory" control samples were highly expressed in cancer-specific,immunological,endocrine and metabolic diseases,and membrane transport functions.This study also annotated the FEGG pathway and found that there were 42 common functions in 26 samples.Among the anaerobic phenotypes,the cholestatoma group in the middle ear had the highest abundance,mainly due to Bacteroidetes and Firmicutes.Among the mobile element content phenotypes,the highest abundance was found in the cholestatoma group of the middle ear,mainly due to Bacteroidetes.In the facultyanaerobic phenotypes,the highest relative abundance was found in the "relatively non-inflammatory state" group,mainly due to Firmicutes.Among the gram-positive phenotypes,the "relatively non-inflammatory state" group was the most abundant.In Pathogenic phenotype,middle ear cholesteatoma group Bacteroidetes expressed abundance is significantly larger than the other two groups,"relatively non-inflammatory state" group Firmicutes expressed abundance is significantly larger than the other two groups.Conclusions:1.Changes in staphylococcus abundance may be diagnostic and prognostic biomarkers in patients with chronic suppurative otitis media(active stage)and cholestatoma of the middle ear.2.Sample flora diversity "state" relative absence of inflammation group and chronic suppurative otitis media(activity)there are differences between the group and middle ear cholesteatoma group,middle ear cholesteatoma group is "relatively no inflammatory state" colony diversity is bigger,chronic suppurative otitis media(activity)colony in the "state" relative absence of inflammation group diversity.3.Compared with the"relatively non-inflammatory state group",the following bacterial communities in the group of chronic purulent otitis media(active stage)and middle ear cholestatoma were decreased,including Bacillus,Staphylococci,WS,Cloacimonadaceae,and Trichoderma,suggesting that these bacterial communities may be used as biomarkers for predicting microenvironmental disorders in the middle ear.4.This study also successfully identified some microflora from patients with chronic suppurative otitis media(active stage),such as:Phylum Lycophyta,class β-Proteobacteria.FamilyXI and W5053 were identified in patients with middle ear cholesteatoma.The relationship between the above bacteria and chronic otitis media(active stage)and middle ear cholesteatoma has not been reported,which is expected to provide a new target and direction for future research on the role and mechanism of middle ear bacteria in the pathogenesis of chronic otitis media(active stage)and middle ear cholesteatoma.5.The pathogenicity of middle ear cholesteatoma may be related to Bacteroidetes. |
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