WAN Youdi,WAN Yan,CHEN Yafei,et al.Effects of antibiotics on gut microbes,metabolites and barrier structure in mice infected with influenza virus[J].Journal of Nanjing Agricultural University,2020,43(6):1056-1062.[doi:10.7685/jnau.201909048]





Effects of antibiotics on gut microbes,metabolites and barrier structure in mice infected with influenza virus
万有娣 万艳 陈雅飞 朱梦洁 雷治海 苏娟
南京农业大学动物医学院动物神经生物学实验室, 江苏 南京 210095
WAN Youdi WAN Yan CHEN Yafei ZHU Mengjie LEI Zhihai SU Juan
Animal Neurobiology Laboratory, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
influenza virusantibioticsintestinal microfloramicrobial metabolismtight junction
[目的] 本文旨在研究流感感染小鼠使用抗生素后肠道微生物、代谢物及肠道紧密连接蛋白的变化。[方法] 30只雄性BALB/c小鼠随机分为3组:对照组、感染组和抗生素组。感染组和抗生素组同时感染流感病毒,抗生素组感染1 d后饮水中添加组合抗生素,3组小鼠感染9 d后取样。16S rRNA基因测序后进行微生物组学分析;用气相色谱-飞行时间质谱(GC-TOF/MS)法进行代谢组学分析;用免疫组化法和免疫印迹法进行结肠紧密连接蛋白的分析。[结果] 与对照组相比,感染组微生物多样性指数(Chao1、Shannon、Simpson)、细菌丰度和代谢物主成分分析(PCA)均无显著差异。与感染组相比,抗生素组微生物多样性指数均极显著下降(P<0.01)。拟杆菌门和变形菌门相对丰度极显著增加(P<0.01),厚壁菌门相对丰度极显著降低(P<0.001)。抗生素组与对照组在PCA中完全分离,有机酸代谢与筛选出的差异微生物具有较强相关性。感染组和抗生素组的紧密连接蛋白(Claudin-1、Occludin)的表达量均极显著降低(P<0.01),抗生素组Claudin-1的表达量与感染组相比极显著降低(P<0.001)。[结论] 抗生素降低肠道微生物的多样性,增加致病菌的侵入,减少有益菌的繁殖,加剧流感诱导的肠道菌群失调,减少有机酸的产生,导致更严重的肠道屏障功能损伤。
[Objectives] The paper aimed to study the changes of intestinal microorganisms,metabolites and tight junction proteins in mice infected with influenza after antibiotics was given.[Methods] Thirty BALB/c male mice were randomly divided into three groups:the control group,the virus-infected group and the antibiotic group. Virus-infected group and antibiotic group were infected with influenza virus simultaneously. One day after infection,mice of antibiotic group were treated with combinative antibiotics by drinking water,and samples were collected at 9 days post infection. Using 16S rRNA gene sequencing and gas chromatography-time of flight mass spectrometry(GC-TOF/MS) was to analyze microbiome and metabonomics of microbiota. The expression level of tight junction proteins was detected by immunohistochemistry and western blot.[Results] Compared with control group,there were no significant differences in microbial diversity indexes,phylum level and principle components analysis(PCA) of virus-infected group. However,microbial diversity indexes(Chao1,Shannon,Simpson) were significantly reduced(P<0.01),and the abundance of Bacteroidetes and Proteobacteria showed a significant increase(P<0.01) together with a remarkable decline(P<0.001) in Firmicutes of antibiotic group when compared with the virus-infected group. The antibiotic group was completely separated from the control group in PCA,metabolites related to organic acids had strong correlations with intestine microorganisms that were screened from antibiotic group. The level of tight junction proteins(Claudin-1,Occludin) expression significantly decreased(P<0.01) in virus-infected group and antibiotic group,and the expression of Claudin-1 in antibiotic group was significantly reduced(P<0.001) compared with virus-infected group.[Conclusions] Antibiotics treatment depleted the diversity of intestinal microbial flora,increased the opportunity for pathogenic bacteria invasion and reduced the colonization of probiotics,which exacerbated the microbial communities dysbiosis,decreased the production of organic acids and caused more serious impairment of intestinal barrier function.


[1] Baden L R,Drazen J M,Kritek P A,et al. H1N1 influenza adisease:information for health professionals[J]. New England Journal of Medicine,2009,360(25):2666-2667.
[2] Dilantika C,Sedyaningsih E R,Kasper M R,et al. Influenza virus infection among pediatric patients reporting diarrhea and influenza-like illness[J]. BMC Infectious Diseases,2010,10:3.
[3] Buffie C G,Jarchum I,Equinda M,et al. Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis[J]. Infection and Immunity,2012,80(1):62-73.
[4] Deriu E,Boxx G M,He X S,et al. Influenza virus affects intestinal microbiota and secondary Salmonella infection in the gut through type Ⅰ interferons[J]. PLoS Pathogens,2016,12(5):e1005572.
[5] Winter S E,Winter M G,Xavier M N,et al. Host-derived nitrate boosts growth of E.coli in the inflamed gut[J]. Science,2013,339(6120):708-711.
[6] Tremaroli V,B?ckhed F. Functional interactions between the gut microbiota and host metabolism[J]. Nature,2012,489(7415):242-249.
[7] Wang J,Li F Q,Wei H M,et al. Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated Th17 cell-dependent inflammation[J]. Journal of Experimental Medicine,2014,211(13):2683.
[8] Fachi J L,de Souza-Felipe J,Pral L P,et al. Butyrate protects mice from Clostridium difficile-induced colitis through an HIF-1-dependent mechanism[J]. Cell Reports,2019,27(3):750-761.
[9] Miao W,Wu X J,Wang K,et al. Sodium butyrate promotes reassembly of tight junctions in caco-2 monolayers involving inhibition of MLCK/MLC2 pathway and phosphorylation of PKCβ2[J]. International Journal of Molecular Sciences,2016,17(10):1696.
[10] Li H X,Liu X L,Chen F Y,et al. Avian influenza virus subtype H9N2 affects intestinal microbiota,barrier structure injury,and inflammatory intestinal disease in the chicken ileum[J]. Viruses,2018,10(5):270.
[11] Cheng S S,Ma X,Geng S J,et al. Fecal microbiota transplantation beneficially regulates intestinal mucosal autophagy and alleviates gut barrier injury[J]. mSystems,2018,3(5):137-154.
[12] Rakoff-Nahoum S,Paglino J,Eslami-Varzaneh F,et al. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis[J]. Cell,2004,118(2):229-241.
[13] 张杰,张晓鹏,李鹏高,等. 小鼠粪便细菌基因组DNA提取方法比较[J]. 实验技术与管理,2017,34(12):50-53,57. Zhang J,Zhang X P,Li P G,et al. Comparison of methods for extracting bacterial genomic DNA from mouse feces[J]. Experimental Technology and Management,2017,34(12):50-53,57(in Chinese with English abstract).
[14] Sun H Z,Wang D M,Wang B,et al. Metabolomics offour biofluids from dairy cows:potential biomarkers for milk production and quality[J]. Journal of Proteome Research,2015,14(2):1287-1298.
[15] Zhang Y,Hou Y L,Wang X Y,et al. The effects of kisspeptin-10 on serum metabolism and myocardium in rats[J]. PLoS One,2017,12(7):e0179164.
[16] Wu S,Jiang Z Y,Sun Y F,et al. Microbiota regulates the TLR7 signaling pathway against respiratory tract influenza A virus infection[J]. Current Microbiology,2013,67(4):414-422.
[17] Brugiroux S,Beutler M,Pfann C,et al. Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium[J]. Nature Microbiology,2017,2(2):16215.
[18] Singh R,Chandrashekharappa S,Bodduluri S R,et al. Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway[J]. Nature Communications,2019,10:89.
[19] Gonzalez A,Krieg R,Massey H D,et al. Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression[J]. Nephrology Dialysis Transplantation,2019,34(5):783-794.
[20] Samuelson D R,Welsh D A,Shellito J E. Regulation of lung immunity and host defense by the intestinal microbiota[J]. Frontiers in Microbiology,2015,6:1085.
[21] Hill C,Guarner F,Reid G,et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic[J]. Nature Reviews Gastroenterology & Hepatology,2014,11(8):506-514.
[22] Landy J,Ronde E,English N,et al. Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer[J]. World Journal of Gastroenterology,2016,22(11):3117.
[23] Schulzke J D,Ploeger S,Amasheh M,et al. Epithelial tight junctions in intestinal inflammation[J]. Molecular Structure and Function of the Tight Junction:From Basic Mechanisms to Clinical Manifestations,2009,1165:294.
[24] Zihni C,Mills C,Matter K,et al. Tight junctions:from simple barriers to multifunctional molecular gates[J]. Nature Reviews:Molecular Cell Biology,2016,17(9):564-580.
[25] Furuse M,Hirase T,Itoh M,et al. Occludin:a novel integral membrane protein localizing at tight junctions[J]. The Journal of Cell Biology,1994,123:1777-1788.
[26] Furuse M,Fujita K,Hiiragi T,et al. Claudin-1 and -2:novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin[J]. Journal of Cell Biology,1998,141(7):1539-1550.
[27] Gumbiner B,Lowenkopf T,Apatira D. Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1[J]. Proc Natl Acad Sci USA,1991,88(8):3460-3464.
[28] Yildiz S,Mazel-Sanchez B,Kandasamy M,et al. Influenza A virus infection impacts systemic microbiota dynamics and causes quantitative enteric dysbiosis[J]. Microbiome,2018,6:9.


 Han Chong,Xu Weiyan,Du Nianxing.ORTHOMYXOVIRUSES AND PARAMYXOVIRUSES ISOLATED FROM APPARENTLY HEALTHY DUCKS[J].Journal of Nanjing Agricultural University,1982,5(6):87.[doi:10.7685/j.issn.1000-2030.1982.02.011]
 WEI Qingtian,LI Pinghua,WANG Han,et al.Effect of dietary Enterococcus faecalis replacing of antibiotic on growth performance,diarrhea rate,humoral immunity and intestinal microflora of nursery pigs[J].Journal of Nanjing Agricultural University,2014,37(6):143.[doi:10.7685/j.issn.1000-2030.2014.06.021]
 CUI Yanan,ZHANG Xuhui,LIU Xiaoyu,et al.Effects of different swine manure treatments on growth, yield and quality of cabbage[J].Journal of Nanjing Agricultural University,2017,40(6):281.[doi:10.7685/jnau.201604036]
 XIE Wanying,ZOU Xi.Arsenite oxidation by phyllosphere bacterial community of duckweed and the effect of antibiotics on this process[J].Journal of Nanjing Agricultural University,2020,43(6):667.[doi:10.7685/jnau.201906059]


更新日期/Last Update: 1900-01-01