【作者】 杨帆；

【导师】 周冬生；

【作者基本信息】 福建农林大学 ， 生物与医药硕士（专业学位）， 2025， 硕士

【摘要】 阴沟肠杆菌复合群（Enterobacter cloacae Complex,ECC）是革兰氏阴性菌,属于肠杆菌科,广泛分布在自然界中。作为院内感染中最常见的感染源之一,它通常引起呼吸道感染、尿路感染和血液感染等多种感染疾病。ECC中包含多个种群,其中最常见的是霍氏肠杆菌和阴沟肠杆菌,其次还有阿氏肠杆菌等菌种。其种群的多样性导致了产生耐药性的原因复杂多样。可移动遗传元件（Mobile Ge-netic Elements,MGEs）可以作为各类抗生素耐药基因（Antibiotic Resistance Genes,ARGs）的主要载体,通过水平基因转移（Horizontal Gene Transfer,HGT）这一途径使细菌耐药性广泛传播和迅速积累。碳青霉烯酶基因容易以MGEs为载体存在于ECC中形成耐碳青霉烯阴沟肠杆菌复合群（Carbapenem-resistant Entero-bacter cloacae Complex,CRECC）。另一方面,ECC具有多重耐药（Multiple Drug Resistance,MDR）的特性,这又为粘菌素耐药基因（mcr）提供了温床,导致预防和控制临床上多重耐药细菌的感染及传播更为困难。面对目前这种严峻的耐药形势,我们亟需深入探究多种耐药基因间的相关性,以及携带多种耐药基因的MGEs在遗传进化中的特征和规律。本研究收集了来自中国10个省份的168株ECC菌株,并进行大规模二代测序（Next-Generation Sequencing,NGS）分析,经过初步耐药基因预测、药物敏感性实验筛查和PCR验证,筛选出45株可能携带多种抗生素耐药基因的CRECC菌株进行三代测序（Third-Generation Sequencing,TGS）。根据三代测序结果,进行进一步的全基因组序列筛查、核定菌种、分析耐药基因及其所处的耐药环境。然后从Gen Bank下载同类型的质粒,并与本实验室筛选出的质粒进行详细的序列比较和遗传结构剖析,再通过多种绘图工具将其结构特征进行可视化处理。最后通过接合转移实验验证多重耐药质粒的转移能力,同时利用产酶实验、药物敏感实验来验证供体菌、受体菌和接合子的耐药表型。对收集的菌株进行表型验证及物种鉴定后,共计纳入19株多重耐药ECC菌株。经过平均核苷酸一致性（Average Nucleotide Identity,ANI）比对,核定这些菌株均属于阴沟肠杆菌复合群,其中大部分为霍氏肠杆菌。经过多位点序列分型（Multilocus Sequence Typing,MLST）,共检测到11种不同的STs。其中ST93、ST78和ST114是国际上公认的高危克隆。在纳入分析的菌株中,至少鉴定出57个耐药基因,共涉及12种不同类型的抗生素或重金属相关的耐药。本研究对11个同时携带mcr基因和金属-β-内酰胺酶（Metallo-β-Lactamase,MBL）基因的质粒进行了分型和详细的结构对比,结果发现其中10个质粒的主要复制子类型为Inc HI2型,且骨架结构较为保守,外源插入区丰富多样,可同时携带多个不同种类ARGs。本研究共涉及53个外源遗传元件（Accessory Genetic Element,AGEs）,经过对基因组序列的详细注释分析,分成6组不同的类型:12个Tn1696相关区域;8个携带bla _IMP的整合子;3个外源插入区;3个携带bla _NDM的转座子;24个mcr-9.1核心遗传结构;3个mcr-10.1核心遗传结构。通过对这些AGEs的比较基因组学分析发现,随着插入元件的插入或同源重组等事件的发生,AGEs逐步进化成复杂的镶嵌结构,在种内和种间传播。接合转移实验显示,同时携带mcr和MBL基因的质粒可以从供体菌转移到受体菌中。综上所述,bla _IMP、bla _NDM和mcr基因主要存在于整合子、转座子和基因盒等MGEs中,并通过一系列转移机制整合到质粒中,有效地促进了耐药基因在细菌内或细菌间的积累和传播。本研究为ECC中编码mcr和MBL基因的MGEs遗传多样性与传播广泛性提供了更深入的认识。这些MGEs在传播过程中通过同源重组或转位进行修饰,进化出复杂的嵌套结构,促进了抗生素耐药性的广泛传播,增强了ECC在多种抗生素压力下的适应性。本研究为后续探究ECC的耐药机制、流行病学特征以及临床感染性疾病的诊断和治疗用药问题提供了分子理论基础,为减缓粘菌素和碳青霉烯酶的耐药性传播提供了重要的遗传学依据。更多还原

【Abstract】 Enterobacter cloacae Complex（ECC）,a Gram-negative bacterium from the En-terobacteriaceae family,commonly found in various environments.It is a frequent cause of hospital-acquired infections,leading to a variety of conditions such as respir-atory,urinary tract,and bloodstream infections.The ECC includes several strains,with Enterobacter hormaechei and Enterobacter cloacae being the most prevalent,followed by species such as Enterobacter asburiae.and others.The diversity among these strains contributes to the complexity of antibiotic resistance.Mobile genetic elements（MGEs）serve as primary vehicles for antibiotic resistance genes（ARGs）,facilitating the rapid spread of resistance through horizontal gene transfer（HGT）.Carbapenemase genes are often found in ECC,resulting in the emergence of Carbapenem-resistant Enterobacter cloacae Complex（CRECC）.Additionally,ECC exhibits multiple drug resistance（MDR）,which fosters the development of the colistin resistance gene（mcr）,complicat-ing efforts to control and prevent infections caused by MDR bacteria in clinical settings.Given the critical nature of this drug resistance issue,there is a urgent need to investi-gate the relationships among multidrug resistance genes and the genetic evolutionary characteristics of MGEs carrying these genes.In this research,we gathered 168 strains from 10 provinces in China between2012-2021 and conducted extensive next-generation sequencing（NGS）analysis.After initial resistance gene predictions,drug susceptibility testing,and PCR validation,45ECC strains were identified as potential carriers of multidrug-resistant genes.We then performed third-generation sequencing（TGS）.Following TGS results,we conducted whole-genome sequencing,strain verification,and analysis of resistance genes and their associated environments,ultimately identifying Enterobacter cloacae strains that carried both carbapenemase and mcr genes for further analysis.We also screened for plasmids of the same type from the Gen Bank,conducted detailed sequence compari-sons and genetic structure analyses with those from our laboratory,and visualized their structural features using various tools.Finally,we confirmed the transferability of mul-tidrug-resistant plasmids through splicing transfer experiments,and we used enzyme-producing and drug sensitivity tests to validate the drug-resistant phenotypes of donor,acceptor,and splicer bacteria.After phenotypic validation and species identification of the collected strains,a total of 19 strains of multidrug-resistant Enterobacter cloacae complex group were in-cluded.After average nucleotide identity（ANI）comparison,it was approved that all these strains were Enterobacter cloacae complex,most of which were E.hor-maechei,and the rest were E.asburiae,.After MLST typing,a total of 11 different STs were detected.Of these,ST93,ST78,and ST114 were internationally recognized as high-risk clones.Among these 11 plasmids included in the analysis,at least 57 re-sistance genes were identified,involving a total of 12 different types of antibiotic-or heavy metal-related resistance.In this study,the 11 plasmids carrying both mcr genes and metallo-β-lactamase（MBL）were typed and subjected to detailed structural com-parisons,and it was found that 10 of these plasmids had the major replicon type of Inc HI2 and had a relatively conserved backbone structure,with abundant and varied exogenous insertion regions,and could simultaneously carry several different types of antimicrobial resistance gene（ARG）In this study,a total of 53 AGEs were involved,which were classified into 6 different groups after detailed annotation and analysis of the genome sequences:12 Tn1696-associated regions,8 bla _IMP-carrying integrons,3exogenous insertion regions,3 bla _NDM-carrying transposons,24 mcr-9.1 core genetic structures,and 3 mcr-10.1 core genetic structures.Comparative genomic analyses of these AGEs revealed that with events such as the insertion of insertion elements or ho-mologous recombination,the AGEs gradually evolved into complex mosaic structures that propagated within and between species.Splice transfer experiments showed that plasmids carrying both the mcr gene and MBL could be transferred from donor bacteria to recipient bacteria.In conclusion,bla _IMP,bla _NDM and mcr genes are predominantly present in MGEs such as integrons and transposons,and are integrated into plasmids through a series of transfer mechanisms,which effectively contribute to the accumulation and dissemina-tion of drug resistance genes within or among bacteria.This study provides a deeper understanding of the genetic diversity and wide spread of MGEs encoding mcr and MBL genes in the Enterobacter cloacae complex.These MGEs were modified by ho-mologous recombination or transposition during transmission,evolving complex nested structures that facilitated the widespread spread of antibiotic resistance and en-hanced the adaptation of the Enterobacter cloacae complex under the pressure of mul-tiple antibiotics.This study provides a molecular theoretical basis for the subsequent investigation of the resistance mechanism and epidemiological characteristics of the Enterobacter cloacae complex,as well as the diagnosis and therapeutic use of clinical infectious diseases,and provides an important genetic basis for slowing down the spread of resistance to mucins and carbapenemases.更多还原