【作者】 王晓明；

【导师】 赵建宏；

【作者基本信息】 河北医科大学 ， 临床检验诊断学， 2018， 硕士

【摘要】 第一部分婴儿肠道艰难梭菌定植情况的动态变化目的:艰难梭菌(Clostridium difficile,CD)是一种专性厌氧的革兰阳性芽孢杆菌,一般认为是人体肠道中的正常菌群,主要通过粪-口途径进行传播。艰难梭菌在婴儿肠道能够长期无症状定植,不同国家地区婴儿艰难梭菌定植率差异较大,且影响婴儿艰难梭菌定植的因素不尽相同。目前关于河北省石家庄地区婴儿艰难梭菌的定植情况尚缺乏相关研究,本研究对河北省石家庄地区婴儿艰难梭菌的定植情况进行系统跟踪调查以了解婴儿肠道艰难梭菌定植的动态变化过程及其影响因素。方法:1收集2016年12月至2018年3月间,河北省石家庄地区的34个婴儿的粪便标本共计538份,分别在每个婴儿出生后24小时、7天、14天、21天、1月、2月、3月、4月、5月、6月、7月、8月、9月、10月、11月、12月龄共计16个时间点留取婴儿新鲜粪便标本,同时记录婴儿的基本资料。2采用富集芽孢法对收集的所有粪便标本进行艰难梭菌的分离、培养和鉴定,检测毒素A、B,并对分离得到的菌株进行PCR-核糖体分型。3用琼脂稀释法对分离得到的艰难梭菌进行药物敏感试验,共选择11种抗生素(替加环素、甲硝唑、万古霉素、非达霉素、左氧氟沙星、环丙沙星、头孢曲松、美罗培南、克林霉素、四环素、氯霉素),分别测定最低抑菌浓度(MIC)值。结果:1.在34名婴儿中,有27名婴儿肠道内检测出有艰难梭菌定植;538份粪便标本中有145份检测出有艰难梭菌定植,定植率为27.0%(145/538);分离得到的145株艰难梭菌,其中90株为产毒株(A+B+),产毒株比例为62.1%(90/145)。2.145株艰难梭菌分属于17个核糖体型别,其中HB2所占比最多为16.6%,其次是HB1(11.0%)、HB6(11.0%)和HB12(10.3%)。在产毒艰难梭菌的核糖体分型中,HB1(17.8%)和HB6(17.8%)占比最多;而非产毒艰难梭菌的核糖体分型中,HB2(43.6%)占比最多。3.艰难梭菌对甲硝唑、万古霉素、非达霉素、替加环素和美罗培南高度敏感,没有出现耐药菌株;对克林霉素、环丙沙星、头孢曲松的耐药率较高,耐药率分别是96.3%,71.6%,25%;对氯霉素、四环素、左氧氟沙星的耐药率较低,分别是11.3%,3.7%,2.5%。结论:本次研究中河北省石家庄地区婴儿艰难梭菌定植率是27.0%,其中产毒株比例是62.1%。婴儿艰难梭菌的定植率随月龄增加有上升的趋势。分离得到的艰难梭菌菌株分属于17种核糖体型别,其中HB1和HB6在产毒艰难梭菌中占比最多,HB2在非产毒艰难梭菌中是优势型别。分离得到的艰难梭菌对美罗培南高度敏感,对左氧氟沙星和四环素的耐药率相对较低。第二部分艰难梭菌定植对6月龄婴儿肠道菌群的影响目的:婴儿期是肠道菌群建立的关键时期,并且婴儿艰难梭菌定植与肠道菌群之间存在一定关系。然而,目前关于河北省石家庄地区婴儿艰难梭菌定植对其肠道菌群的影响尚缺乏相关研究。本研究中,我们采用16S rRNA基因高通量测序技术对6月龄婴儿肠道菌群进行分析,进而了解艰难梭菌定植对6月龄婴儿肠道菌群是否有影响。方法:1选取本实验第一部分中6月龄之内有艰难梭菌定植的婴儿10名,6月龄之内无艰难梭菌定植的婴儿10名,并收集此20名婴儿的6月龄的粪便标本共计20份。同时记录婴儿的基本资料。2采用富集芽孢法对收集的20份粪便标本进行艰难梭菌的分离、培养和鉴定,并检测毒素A、B。3对粪便基因组DNA的16S rRNA基因V3和V4区进行测序分析。结果:1.通过16S rRNA基因高通量测序技术分析显示,婴儿在6月龄之内有无艰难梭菌定植,其6月龄婴儿的肠道菌群差异无统计学意义。2.物种分类分析显示,在门水平上,6月龄婴儿肠道菌群主要是变形菌门、厚壁菌门、放线菌门和拟杆菌门(总占比接近100%);有艰难梭菌定植的婴儿,其肠道菌群中厚壁菌门最多,无艰难梭菌定植的婴儿,其肠道菌群中变形菌门最多。在属水平上,6月龄婴儿肠道菌群主要是埃希氏菌属、双歧杆菌属和韦荣氏球菌属(总占比接近70%);有艰难梭菌定植的婴儿,其肠道菌群中韦荣氏球菌属最多,而无艰难梭菌定植的婴儿,其肠道菌群中埃希氏菌属最多。3.菌群丰度差异分析显示,在有艰难梭菌定植的婴儿中,有显著作用的微生物类群是:梭菌纲、梭菌目、毛螺菌科、瘤胃球菌科、梭菌属、毛螺菌属、解黄酮菌属;在无艰难梭菌定植的婴儿中,有显著作用的微生物类群是:巴斯德菌目、巴斯德菌科、嗜血杆菌属。结论:婴儿有无艰难梭菌定植对6月龄婴儿肠道菌群影响不大。更多还原

【Abstract】 Part one Follow-up survey of Clostridium difficile colonization in infantsObjective: Clostridium difficile(CD)is an obligate anaerobic grampositive bacillus generally considered to be the normal flora of the human intestine.The bacteria are mainly transmitted through the faecal-oral route.C.difficile could be asymptomatically colonized in infants for a long period of time,however the cause and influencing factors of colonization are still far more limited.We conducted a 1-year follow-up survey in infants in Shijiazhuang City,Hebei Province to understand the dynamic changes of C.difficile colonization and the influencing factors.Methods:1 A total of 538 stool specimens from 34 infants were collected from December 2016 to March 2018 in Shijiazhuang City,Hebei Province.Fresh stool specimens were collected at 16 points.Basic information was recorded.2 The enriched spore method was used to separate,culture and identify for C.difficile.C.difficile isolates were typed by PCR-ribotyping and analyzed for the presence of toxin genes.3 Antimicrobial susceptibility testing was performed on 11 antibiotics involved levofloxacin,ciprofloxacin,ceftriaxone,meropenem,clindamycin,tetracycline,chloramphenicol,metronidazole,vancomycin,fidaxomicin and tigecycline for the minimum inhibitory concentration(MIC).Results:1.27 of the 34 enrolled infants acquired C.difficile during the first year of life.145 fecal specimens contained C.difficile of total 538 specimens(27.0%,145/538),and 90 were toxigenic strains(62.1%,90/145).2.145 strains were divided into 17 ribotypes.HB2 accounted for thelargest proportion of all ribotypes,accounting for 16.6% of all strains,followed by HB1(11.0%),HB6(11.0%),HB12(10.3%).HB1 and HB6 accounted for the most in toxigenic strains,HB2 accounted for the most in non-toxigenic strains.3.All isolates were highly susceptible to metronidazole,vancomycin,fidaxomicin,tigecycline and meropenem.The resistance rate of clindamycin,ciprofloxacin,and ceftriaxone were 96.3%,71.6%,and 25%,respectively.The resistance rates of chloramphenicol(11.3%),tetracycline(3.7%),and levofloxacin(2.5%)were low,respectively.Conclusions: The colonization rate of C.difficile in infants in Shijiazhuang City,Hebei Province was 27.0%,and toxigenic C.difficile was62.1%.The colonization rate of C.difficile in infants increased with age.17 ribotypes were identified,HB1 and HB6 accounted for the most in toxigenic C.difficile while HB2 accounted for the most in non-toxigenic C.difficile.The C.difficile isolated from infants were highly susceptible to meropenem,and the resistance rates of tetracycline and levofloxacin were relatively low.Part two Effects of Clostridium difficile colonization on the intestinal flora in 6-month-old infantsObjective: Infancy is a critical period for the establishment of intestinal flora,and there is a certain relationship between C.difficile colonization and intestinal flora of infants.However,at present,the effects of C.difficile colonization on the intestinal flora of infants in Shijiazhuang City,Hebei Province is still lacking.In this study,we used 16 S rRNA gene highthroughput sequencing technology to analyze the intestinal flora of 6-monthold infants,and then to understand whether C.difficile colonization has an effect on the intestinal flora in 6-month-old infants.Methods:1 10 infants with C.difficile colonization and 10 infants without C.difficile colonization within 6 months of age in the first part of the experiment were included.Fresh stool specimens were collected at 6 months of age.Basicinformation was recorded.2 The enriched spore method was used to separate,culture and identify for C.difficile.C.difficile isolates were analyzed for the presence of toxin genes.3 Fecal genomic DNA was analyzed by V3 and V4 regions of 16 S rRNA gene results.Results:1.C.difficile colonization had no significant effect on the composition of intestinal flora in 6-month-old infants.2.At the phylum level,the intestinal flora were mainly consisted of Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes.Firmicutes was the most portion in infants with C.difficile colonization,while Proteobacteria was the most portion in infants without C.difficile colonization.At the genus level,Escherichia/Shigella,Bifidobacterium and Veillonella accounted nearly70%.Veillonella was the most portion in infants with C.difficile colonization,while Escherichia/Shigella was the most portion in infants without C.difficile colonization.3.The significant microbial groups were Clostridiales,Clostridia,Lachnospiraceae,Lachnospiracea_incertae_sedis,Clostridium_XlVa,Ruminoc occaceae,and Flavonifractor in group with C.difficile colonization,were Haemophilus,Pasteurellales and Pasteurellaceae in group without C.difficile colonization.Conclusions: The presence or absence of C.difficile colonization had little effect on the intestinal flora in 6-month-old infants.更多还原