【作者】 刘颖；

【导师】 宗志勇；

【作者基本信息】 四川大学 ， 内科学（传染）（专业学位）， 2021， 博士

【摘要】 目的:结合全基因测序结果,通过对华西医院临床分离和肛拭子筛查收集到的碳青霉烯类耐药肺炎克雷伯菌（carbapenem-resistant Klebsiella pneumoniae,CRKP）进行耐药表型、毒力基因、序列类型（sequence type,ST）以及临床特点分析来了解其分子流行病学特点。基于单核苷酸多态性（single nucleotide polymorphism,SNP）对ST11型CRKP构建系统进化树,分析其克隆性流行播散的特点,鉴定出流行播散的优势克隆分支,通过比较基因组学探讨其特异性基因、特异性SNP和分化时间。以比较基因组学分析结果为指向,对优势克隆和亚优势克隆的代表菌株在生存表型上进行了包括动物实验在内的一系列对比研究,探讨优势克隆广泛播散的机制。方法:1、收集四川大学华西医院ICU病房自2017年3月27日至2017年8月31日期间肛拭子常规筛查分离和临床标本中分离的CRKP菌株。使用含美罗培南和利奈唑胺的筛选琼脂培养分离纯化,确认碳青霉烯类耐药表型,通过扩增gyr B初步鉴定菌种。采用微量肉汤稀释法进行体外药敏试验,检测12种常见抗菌药物的最低抑菌浓度（MICs）。使用二代Illumina Hi Seq X10测序平台进行全基因组测序,BBMap v35.85对测序短序列质量控制,Shovill v1.1.0进行细菌基因组拼接及质控,Prokka v1.14.5进行功能注释,Abricate v0.9.2寻找耐药基因、毒力基因和质粒类型,通过MLST数据库进行ST分型。对临床资料进行回顾性分析。2、基于二代Illumina全基因组测序结果,对ST11型CRKP使用Snippy v4.5.2鉴定SNP,Snp-dists v0.6.3建立SNP矩阵,Gubbins v2.4.1剔除重组序列,RAx ML v8.2.12构建基于最大似然法的系统发育树等一系列生物信息学分析,了解ST11型是否存在多个克隆,是否有特定的克隆呈现明显的传播优势。纳入样本采集时间,经Bact Dating v1.0演算系统发育树上菌株数量最多的群体（优势克隆）和菌株数量次于优势克隆的群体（亚优势克隆）分支节点的发生时间,从而推断群体的起源时间和不同类群的分化时间。联合Roary v3.13.0和Scoary v1.6.16寻找优势克隆的特异性基因。联合Snippy v4.5.2和Scoary v1.6.16寻找优势克隆特异性SNP。使用Swiss-model同源建模、I-TASSER折叠识别预测蛋白三维结构,计算SNP改变前后蛋白分子结构的偏移。3、基于比较基因组学分析结果,通过透射电镜直接观察、半固体琼脂动力试验了解优势克隆和亚优势克隆代表菌株菌毛形态和功能。在无菌聚氯乙烯（PVC）材质的96孔板中使用结晶紫染色、冰乙酸溶色方法测定生物膜形成量,以了解菌株在非生物表面形成生物膜的能力。通过健康捐赠者人血清与菌悬液混合培养计数方法,评价菌株对血清抗性和敏感等级。使用大蜡螟幼虫模型测定菌株毒力,了解优势克隆和亚优势克隆菌株毒力和播散能力的关系。通过绘制菌株分别在需氧或厌氧条件下铁丰富或铁缺乏的LB培养基中的生长曲线,观察菌株在不同条件下的生长情况,以了解菌株在不同条件下利用有限物质进行生长的能力。通过在需氧或厌氧条件下铁丰富或铁缺乏的LB培养基中对优势克隆和亚优势克隆的代表菌株共培养,了解两菌在共生环境中竞争利用物质进行生长的能力。将菌株单独菌悬液灌胃已建立的小鼠肠道微生态功能紊乱模型,了解菌株在肠道中生长情况。再将优势克隆和亚优势克隆代表菌株的混合菌悬液灌胃小鼠模型,了解两菌在肠道中共生时是否呈竞争性生长。4、分别对在肠道内共生时表现出明显生长优势或劣势的菌株进行转录组水平测序,使用HTSeq v0.6.1估计基因表达水平,DESeq进行两个比较组合之间的差异表达分析,通过GOseq和KOBAS分析差异表达基因的GO富集和KEGG通路富集。使用RNAiso Plus提取细菌RNA,通过实时荧光定量PCR检测细菌乙醇胺氨裂解酶重链蛋白编码基因eut B和转录调节因子编码基因eut R m RNA水平,验证转录组测序分析中乙醇胺通路上调的结果。配置乙醇胺作为唯一碳源或氮源的改良M9培养基,绘制菌株在培养基中的生长曲线,了解菌株利用乙醇胺作为碳源或氮源提供能量的能力。在乙醇胺作为唯一氮源的培养基中将优势克隆和亚优势克隆的代表菌进行共培养,了解菌株在共生环境中抢占乙醇胺作为氮源提供能量的能力。通过ELISA方法测量小鼠不同状态下粪便中维生素B12的含量,了解体内环境中菌株合成释放或摄入维生素B12的情况。测量菌株在体外不同培养基中培养后维生素B12的含量,了解菌株合成分泌或摄入维生素B12的能力。结果:1、本研究检出CRKP 174株,153株来自于肛拭子,21株来自临床标本,以医院内获得为主。它们全部为多重耐药,97.1%的CRKP携带bla _KPC-2碳青霉烯酶基因。发现1株多粘菌素低水平耐药菌株。所有产KPC酶的CRKP菌株均对头孢他啶-阿维巴坦敏感。本研究中CRKP有88.5%为ST11型,83.7%的CRKP荚膜K抗原类型为KL64型,93.1%的CRKP脂多糖O抗原类型为O2v1型,所有菌株都携带了3型菌毛基因、铁摄取相关的耶尔森毒素（yersiniabactin）基因。2、本研究中CRKP以ST11型为主,基于SNP构建的系统进化树ST11型CRKP分为7个克隆,其中有一个分支菌株量最多,占据了绝对优势。结合临床资料发现,优势克隆菌株首次被采集到的时间晚于亚优势克隆,仍然形成了占优的形势。时间校正后演算的系统进化树也证明菌系祖先分化时间的早晚与是否形成优势克隆并无明显联系。优势克隆平均携带10.8个耐药基因,亚优势克隆平均携带15.8个耐药基因,提示耐药基因携带数量增多与菌株院内传播优势没有相关。优势克隆和亚优势克隆的比较基因组学分析发现,优势克隆缺少了一个长约10.5kb的基因片段,其中包含10个连续的蛋白编码区,分别是fim D、yad V、mlt F、fab G、ghr A、clo R、btu D和三个没有明确基因名称的蛋白编码区,多数与菌毛合成有关,其他蛋白编码区与氧化还原反应和能量代谢有关。在优势克隆中该片段被一个插入序列ISKpn26替代,提示是由于IS随机插入,优势克隆出现时就丢失了这一片段。同时,优势克隆还含有5个特异性的非同义SNP和1个碱基缺失的位点,涉及到的蛋白编码区功能可能与生物膜合成相关。3、优势克隆和亚优势克隆菌株菌毛形态功能、血清抗性无明显差异。在非生物体表现形成生物膜的能力,优势克隆弱于亚优势克隆,说明优势克隆的播散能力并不来自于粘附在物体表面的能力。优势克隆菌株毒力弱于亚优势克隆。在铁缺乏条件下共生时,优势克隆较亚优势克隆略有生长优势。优势克隆菌株和亚优势克隆菌株混合灌胃小鼠后,第3天粪便中亚优势克隆菌株含量基本为0,在竞争中优势克隆菌株表现出明显的生存优势。4、对分离自粪便样本的菌群进行转录组学分析,优势克隆菌株在与亚优势克隆菌株混合灌胃后在小鼠肠道内出现了维生素B12厌氧合成途径、乙醇胺代谢途径和1,2-丙二醇代谢途径通路基因的集中上调（log2差异倍数≥3倍）。乙醇胺氨裂解酶重链蛋白编码基因eut B和转录调节因子编码基因eut R m RNA水平验证了优势克隆菌株乙醇胺代谢通路的上调。两个代表菌株均可利用乙醇胺作为唯一氮源提供能量进行生长,但在乙醇胺作为唯一氮源的培养基中共生时,优势克隆菌株对亚优势克隆菌株表现出适应性优势。与正常小鼠相比,肠道微生态功能紊乱的小鼠粪便中维生素B12含量没有变化,灌胃菌悬液后粪便中维生素B12的含量明显增加,可能来自于细菌在肠道中合成增多,或肠道常驻菌的合成增多。第3天优势克隆菌株灌胃的小鼠粪便中维生素B12含量较亚优势克隆菌株灌胃小鼠明显增多,提示优势克隆菌株合成维生素B12能力比亚优势克隆菌株更强。结论:华西医院CRKP对碳青霉烯类高水平耐药,绝大多数携带bla _KPC-2,以ST11型为主。在ST11型CRKP中发现菌株数量最多的优势克隆。克隆祖先分化时间的早晚与是否形成优势克隆并无明显联系。优势克隆在竞争共生环境中主要通过上调厌氧途径合成维生素B12以及利用乙醇胺能力更强,促使其在肠道中处于竞争优势。相对较低的毒力也有利于感染者长期携带,从而促进播散。维护肠道菌群平衡对于预防肠道定植极为重要,医院感染防控要和抗菌药物科学管理相结合。肠道病原菌利用底物的能力是其在肠道内定植的关键因素,可能为设计预防肠道定植的策略提供新思路。更多还原

【Abstract】 Objectives:To characterize molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae(CRKP)in West China Hospital by their antimicrobial resistance determinants,virulence genes and sequence types(ST)based on the whole genome sequences analysis.To identify characteristics of the transmission of ST11 type CRKP.To examine clonal relatedness of ST11 CRKP isolates based on single nucleotide polymorphisms(SNP).To study mechanisms mediating the successful transmission of the predominant clone using a set of experiments informed by comparative genomics analysis.Method:1.CRKP isolates recovered from clinical samples and rectal swab isolates at ICU of West China Hospital,Sichuan University,between March 27 th and August 31 st in 2017 were collected.After species identification of these strains by sequencing the gyr B gene,the resistant phenotypes for 12 commonly-used antimicrobial agents were tested using the broth microdilution method of the Clinical and Laboratory Standards Institute(CLSI).All of the CRKP isolates were sequenced using the Illumina Hi Seq X10 platform,and the generated raw reads were trimmed by BBMap v35.85,assembled by Shovill v1.1.0 and annotated by Prokka v1.14.5.Antimicrobial resistance genes,virulence genes and plasmid information were then identified using the Abricate v0.9.2 program.Multilocus sequence typing(MLST)was performed using the MLST database.Clinical data were also analyzed.2.SNPs were called by Snippy v4.5.2 after removing recombination detected by Gubbins v2.4.1.Pairwise SNP distance matrix was generated by Snp-dists v0.6.3.A maximum-likelihood phylogenetic tree including all isolates was inferred by RAx ML v8.2.12.ST11 CRKP isolates were assigned to clones based on the SNPs and the phylogenetic tree.By including the collection date of individual isolates,the maximum-likelihood tree was further dated using Bact Dating v1.0.Genes and SNP specific to the predominant clone were identified by Scoary v1.6.16 and Roary v3.13.0 or Snippy v4.5.2.The three-dimension(3D)model of proteins with amino acid substitutions were predicted by the homology modelling web tool Swiss-model and the threading server I-TASSER.3.Fimbriae morphology of the representative strain of the predominant clone and that of the subdominant clone was observed through transmission electron microscope.Cell motility was examined on semi-solid agar.Serum resistance assay was performed by co-incubation with serum donated by health volunteers.Biofilm formation capacity was measured in sterile polyvinyl chloride(PVC)microtiter panel using crystal violet staining and glacial acetic acid dissolution.The Galleria mellonella larva infection model was used to assess the virulence of representative strains.Growth in iron-adjusted nutrient-restricted media was tested.In vitro competition assays between the two representative strains were performed incubated in iron-adjusted media.In vivo competition assay was tested using antimicrobial-treated mice inoculated with bacterial suspensions via oral gavage.Competition advantage was then measured by cell viable counts collected from mice fecal particles.4.Transcriptome sequencing was performed for the strain that showed obvious competition advantage in mice from the co-inoculated mixture.HTSeq v0.6.1 was used to count the number of reads mapped to each gene.Differential expression of two groups was analyzed using the DESeq R package v1.18.0.Gene ontology(GO)enrichment analysis of differentially expressed genes was performed by the GOseq R package.KOBAS software was used to examine the statistical enrichment of differential expression genes in KEGG pathways.RNA used for quantitative reverse transcription polymerase chain reaction(q RT-PCR)was prepared using the RNAiso Plus kit.q RT-PCR was then performed to eut B(encoding ethanolamine ammonia-lyase heavy chain)and eut R gene(encoding HTH-type transcriptional regulator)expression levels.The capacity of the two representative strains to grow with ethanolamine as the sole carbon or nitrogen source was tested on modified M9 medium supplemented with the corresponding substances.Competition assays were performed between the two representative strains co-incubated in modified M9 media with ethanolamine as the sole nitrogen source.The concentration of vitamin B12 in the mice fecal particle after the mice were mouth-fed with different bacterial suspensions for day 1,day 3 and day 7 respectively,were measured by ELISA.Vitamin B12 in the medium incubating different cultures was determined using the above method.Results:1.A total of 174 CRKP isolates were obtained with 153 from rectal swabs and 21 from clinical specimens.All isolates were multi-drug resistance.Most of the isolates carried carbapenemase gene bla KPC-2.One low-level colistin-resistant strain was also found.All KPC-producing CRKP isolates were susceptible to ceftazidime-avibactam,although was ineffective against CRKP producing class B carbapenemase NDM.ST11 was the dominant ST type of CRKP in this study.The major capsular K-antigen type was KL64,and the major lipopolysaccharide O-antigen type was O2v1.As for virulence factors,all isolates carried type 3 fimbriae coding genes and yersiniabactin associated with iron uptake.2.ST11 CRKP in this study could be assigned to seven distinct clones,among which there was a predominant clone comprising most ST11 isolates.A time-calibrated phylogenetic analysis was inferred base on all ST11 CRKP isolates in this study,suggesting that the emerging time of the most recent common ancestor was irrelevant to the formation of predominant clone.An average of 10.8 antimicrobial resistance(AMR)genes were identified from strains belonging to the predominant clone,whereas fewer than the 15.8 on average in those from subdominant clone,the clone comprising the second largest number of isolates.The comparative genomics analysis demonstrated that the predominant clone lacked an approximate 10.5-kb gene fragment,which contained 10 consecutive protein-coding genes including fim D,yad V,mlt F,fab G,ghr A,clo R and btu D and three genes with unknown function.Most of those genes encoding proteins for fimbriae synthesis process,while others were associated with redox and energy metabolism.This fragment was replaced by an ISKpn26 in the predominant clone,implying the defective fragment emerged before divergence between the predominant clone and the subdominant clone.In addition,the predominant clone had 5 specific non-synonymous SNPs and 1 nucleotide deletion,all of which involved in the biofilm synthesis.3.The predominant and subdominant clones displayed no significant difference in fimbriae morphology and function,as well as the ability of serum resistance ability.The capacity of biofilm formation on PVC surface was weaker in predominant clone compared with the other clone,indicating that the dissemination advantage was no relevant to biofilm formation.In addition,the predominant clone exhibited lower virulence than the subdominant clone.The representative strain of the predominant clone had a slightly competitive advantage over the other when co-incubated in the iron-defective medium.Obvious competition advantage fitness was also observed for the representative strain of the predominant clone over that of the subdominant clone in fecal particles from mice received mouth-fed commensal bacterial suspension.4.Transcriptome sequencing of the representative strains in the mono-and co-cultures from mice fecal particles was performed.Differential gene expression analysis of the representative strain of the predominant clone compared to the monoculture control in mice gut revealed four significantly upregulated gene clusters,including type 2 secretion system,vitamin B12 anaerobic synthesis pathway,ethanolamine utilization and 1,2-propanediol utilization pathway.Relative fold change in eut B and eut R confirmed the upregulation at the m RNA level.Both of the tested CRKP strains could use ethanolamine as the sole nitrogen source for growth.While co-cultured in the nutrient-restricting condition with ethanolamine as the sole nitrogen source,the representative strain of the predominant clone showed a slight competition fitness to the other one.Compared with that in mice without antimicrobial treatment,the vitamin B12 concentration in feces of mice with microflora disturbed by antimicrobials increased after gavage of the bacterial suspension.The concentration of vitamin B12 in the mice fecal particle after the mice were mouth-fed with the predominant bacterial suspensions on day 3 was increased compare to those mouth-fed with the subdominant bacterial suspensions,suggesting that the predominant clone had stronger capability to synthesize vitamin B12.Conclusions:CRKP collected from West China Hospital showed high levels resistance to carbapenems,and most of them carry bla KPC-2.ST11 was the dominant ST type spread in the hospital.A predominant clone comprising most ST11 CRKP isolates was identified.The emerging time of the most recent common ancestor of each clone was irrelevant to the emergence of predominant clone.The predominant clone had competition advantage mainly due to the increased vitamin B12 anaerobic synthesis and enhanced ethanolamine utilization in co-culturation.The capacity of utilizing ethanolamine as a nitrogen source conferred a growth advantage to the representative strain of the predominant clone when co-cultured with that of the subdominant clone.Lower virulence might contribute to the long-term carriage of the predominant clone in colonized patients,thereby leading to more successful dissemination.The stable gut microbe community was critical to gut colonization resistance.The prevention and management of hospital infection should be combined with the antimicrobial stewardship(AMS).Our findings suggested that the ability of enteric pathogens to utilize substrates provided an advantage during colonization,which was important for rationally designing future strategies for prevention of gut colonization and decolonization.更多还原