【作者】 李伟；

【导师】 杨保伟；

【作者基本信息】 西北农林科技大学 ， 食品科学与工程， 2021， 博士

【摘要】 多粘菌素是治疗多重耐药革兰氏阴性菌,特别是碳青霉烯耐药肠杆菌科细菌感染的“最后一道防线”。几十年来,多粘菌素在各大洲广泛地应用于预防和治疗动物肠道疾病,在中国、日本、印度和越南等一些亚洲国家被添加在养殖动物的饲料中作为促生长剂使用。由于质粒介导的多粘菌素耐药基因mcr-1的流行,加速了多粘菌素耐药性的传播。导致多粘菌素耐药菌株的存在越来越广泛,为养殖动物健康及食品安全生产带来了极大的隐患。本课题通过研究不同温度和不同介质等因素对mcr-1基因在食源性野生型肠杆菌科细菌之间的水平转移能力的影响,验证mcr-1基因在小鼠肠道内的水平转移情况,以及转移过程中mcr-1基因环境的变化。从基因层面分析了mcr-1基因的转移机制。为寻求减缓或阻断mcr-1基因的快速、广泛传播提供理论依据,为动物养殖及食品安全做出贡献。（1）本研究首先对实验室于2006～2017年间分离的食源性沙门氏菌和大肠杆菌进行了mcr-1阳性细菌的筛查。共检测了1,789株细菌,其中沙门氏菌1,283株,大肠杆菌506株。菌株分离于超市或农贸市场中零售的食品样品（鸡肉、猪肉、牛肉、羊肉、熟食、婴幼儿配方奶粉、饺子和鱼）,食品生产链条（养殖场和屠宰场）以及临床病例样品（门诊中患有食源性疾病病人的粪便）。菌株来自中国北京、上海、河南、陕西、四川、广东、广西、福建和新疆9个地区。mcr-1阳性菌检出率为6.7‰（12/1,789）。2006～2017年间分离的大肠杆菌中mcr-1阳性菌检出率为21.7‰（11/506）,显著高于同期分离的沙门氏菌中mcr-1阳性菌的检出率（0.78‰,1/1,283;P<0.05）。（2）为了验证mcr-1基因能够在细菌之间进行水平转移同时介导低水平的多粘菌素耐药,筛选了9株对多粘菌素B具有耐药性的mcr-1阳性菌作为供体菌（命名为D1～D9）进行接合转移试验。同时,为了探究不同温度与不同介质等因素对mcr-1基因在菌株之间进行水平转移能力的影响,选择鸡肉为代表食物进行以鸡肉为基质的接合转移试验。为了模拟摄入mcr-1阳性菌污染的食物后mcr-1基因在动物肠道中的水平转移情况,以昆明白鼠构建动物模型进行接合转移试验。结果显示,滤膜-培养基条件下mcr-1基因的接合频率为1.57×10^-5～4.60×10^-2,显著高于37℃下mcr-1基因在鸡肉中的接合频率1.32×10^-6～3.85×10^-4。即使在4℃下,mcr-1基因也能在鸡肉中成功的从供体菌转移到受体菌。在小鼠肠道中,mcr-1基因不仅可以转移到灌胃的受体菌中,还能够转移到小鼠肠道菌中。mcr-1基因在小鼠肠道内水平转移对小鼠肠道菌群产生了负面的影响。D6-E.coli C600组中检测到的接合子峰值为1.43×10²CFU/g粪便,显著高于D7-E.coli C600组检测到的接合子峰值0.3×10² CFU/g粪便。在相同的接合转移条件下,质粒的复制子类型是影响接合频率的最重要因素,即Inc HI2型质粒的接合转移能力更强。（3）通过分析携带mcr-1基因的质粒序列,共检测到3种编码mcr-1基因的质粒复制子类型,分别是Inc X4,Inc I2和Inc HI2。进一步分析mcr-1基因环境发现,在Inc X4型质粒中mcr-1基因和上游的插入序列构成IS26-mcr-1-pap2的基因环境,在mcr-1基因水平转移过程中未发生改变。在D2和D4携带的Inc I2质粒型中mcr-1基因和上游的插入序列构成ISApl1-mcr-1-pap2的基因环境。在滤膜-培养基接合过程中,D2中mcr-1基因上游的插入序列ISApl1丢失;在25℃鸡肉中的接合转移过程中,D4中mcr-1基因上游的插入序列ISApl1丢失。在D5携带的Inc I2质粒型中mcr-1基因和上游的插入序列构成ISEcp1-bla_CTX-M-55-mcr-1-pap2的基因环境,在mcr-1基因水平转移过程中未发生改变,是未被报道过的mcr-1基因环境。在Inc HI2型质粒中mcr-1基因和上游的插入序列构成ISApl1-mcr-1-pap2的基因环境,尽管在mcr-1基因水平转移过程中基因环境未发生改变,但是Inc HI2型质粒在细菌繁殖和进化过程中很活跃,容易与其他质粒或基因盒相互作用形成融合质粒。（4）在滤膜-培养基接合过程中鉴定了3个携带mcr-1阳性融合质粒的接合子。D1（R1）1中的p D1（R1）1＿mcr-1F是D1中2个质粒和R1中1个质粒融合形成的Inc HI2质粒;p D1＿MDR中的基因盒IS26-oqx A-oqx B和p D1＿mcr-1中的基因盒IS26-dfr A12-aad A2和IS26-mcr-1水平转移到质粒p R1中,融合质粒与p R1共享相同的基因序列骨架。接合子D2（R5）2中的p D2（R5）2＿mcr-1F是由两个最初存在于D2中的质粒融合形成的一个Inc HI2型质粒;是由p D2＿mcr-1中的基因盒ISApl1-mcr-1-pap2插入p D2＿ami形成的融合质粒,随后转移到了R5中。接合子D6（EC）3中的p D6（EC）3＿mcr-1是D6中p D6＿mcr-1和p D6＿tet（M）通过38 bp的同源区域发生同源重组而形成的,被鉴定为一种新型Inc FIB-Inc HI2杂交质粒。3个融合质粒同时携带mcr-1和其他抗生素耐药基因。质粒融合频率为1.48×10^-3～3.94×10^-3,融合质粒表现出高稳定性,可以将包括mcr-1在内的抗生素耐药基因盒以2.32×10^-4～1.73×10^-3的频率转移到大肠杆菌E.coli C600或J53中。质粒融合事件发生频率较高,机制复杂,融合质粒能够进行稳定遗传并具有较强接合转移能力。更多还原

【Abstract】 Polymyxin is the"last line of defense"for the treatment of multidrug-resistant Gram-negative bacteria,especially Carbapenem-resistance Enterobacteriaceae infections.For decades,polymyxin has been widely used in the prevention and treatment of animal infectious diseases on all continents and has been added to feed as a growth promoter in some Asian countries such as China,Japan,India and Vietnam.The discovery and prevalence of the plasmid-mediated polymyxin resistance gene mcr-1 has accelerated the horizontal transfer and spread of the polymyxin resistance gene.The widespread existence of polymyxin-resistant strains has brought great emergency to the farmed animals health and the food production safety.In this investigation,by clarifying the relevant transmission route of mcr-1 gene in wild-type foodborne Enterobacteriaceae bacteria not only in vitro but in vivo.Explore the influence of factors such as different temperatures and different media on mcr-1 gene transfer ability,as well as the changes in the environmental characteristics of mcr-1 gene during conjugation transfer.Analyze the transfer mechanism of mcr-1 gene from the genetic level.To provide a theoretical basis for seeking to slow down or block the rapid and widespread spread of mcr-1 gene,and reference for food animal breeding and food safety control.（1）We retrospectively investigated the prevalence of mcr-1-positive Salmonella and Escherichia coli isolated from retail food products,food supply chain,and clinical samples across China in the last decade（2006–2017）.Twelve（0.67%）mcr-1-positive strains were identified from 1,789 non-duplicated isolates,including 1,283 Salmonella and 506 E.coli.Eleven（21.7‰）mcr-1-positive E.coli strains were identified from the isolates collected during 2006–2017,which was significantly more prevalent than mcr-1-positive Salmonella in this period（0.78‰,1/1,283;P<0.05）.（2）In order to verify that the mcr-1 gene can transfer horizontally between bacteria and mediate low levels of polymyxin resistance,nine mcr-1-positive isolates resistant to polymyxin B were screened as donors（designated as D1–9）.To explore the influence of factors such as different temperatures and different media on the horizontal transfer ability of mcr-1 gene,chicken was selected as a substrate to carry out a conjugative transfer experiment.And to simulate the horizontal transfer of mcr-1 gene in animal intestines after ingestion mcr-1 positive bacteria contaminated food,an animal model was constructed with Kunming mice for conjugative transfer experiments.In filter-medium conjugation transfer,mcr-1-carrying plasmids in donors were successfully transferred to recipients at frequencies of 1.75×10^-5to 4.6×10^-2 per recipient cell,which was significantly higher than frequencies of conjugation transfer on chicken 1.32×10^-6 to 3.85×10^-4.mcr-1-carrying plasmids in donors were successfully transferred to recipients on chicken even at 4℃.In the mice gut,mcr-1 can not only spread to the recipient bacteria provided by intragastric administration,but also conjugated with intestinal bacteria.The negative effect of mice intestinal flora was showed by the horizontal transfer of mcr-1 gene to mice gut bacteria.In the same conjugative transfer environment,replicon type of plasmids was the most decisive factor that effect the frequencies.The peak number of transconjugants in group D6-E.coli C600with Inc HI2 type plasmid was 1.43×10² CFU/g feces,which was significantly higher than the peak number of transconjugants in group D7-E.coli C600 0.3×102 CFU/g feces.（3）A total of 3 types of plasmid replicators encoding the mcr-1 gene were detected,Inc X4（D1 and D7）,Inc I2（D2,D4 and D5）and Inc HI2（D6 and D9）,respectively.Inc X4-type plasmids carried mcr-1 with the insertion sequence IS26 upstream.The mcr-1 gene cassette IS26-mcr-1-pap2 did not change during the horizontal transfer.The mcr-1 gene cassette ISApl1-mcr-1-pap2 existed in Inc I2-type plasmids carried by D2 and D4.The insertion sequence ISApl1 existed in D2 was lost during filter-medium conjugation transfer and the ISApl1 existed in D4 was also lost during conjugation transfer on chicken in 25℃.Inc I2-type plasmids in D5 carried the mcr-1 gene cassette ISEcp1-bla _CTX-M-55-mcr-1-pap2.The mcr-1 gene cassette ISApl1-mcr-1-pap2 in Inc HI2 type plasmid was stable,which was an unreported mcr-1 genetic environment.The mcr-1 gene cassette ISApl1-mcr-1-pap2 in Inc HI2 type plasmid was stable.Inc HI2 was very active in the process of bacterial reproduction and evolution and it was easy to interact with other plasmids or gene cassettes to form a fusion plasmid.（4）Three mcr-1-bearing fusion plasmids were identified in the transconjugants.Sequence analysis showed that p D1（R1）1＿mcr-1F in the transconjugant D1（R1）1 was an Inc HI2 plasmid formed upon the fusion of two plasmids in D1 and one plasmid in R1;the gene cassettes IS26–oqx A–oqx B in p D1＿MDR and IS26–dfr A12–aad A2 and IS26–mcr-1 in p D1＿mcr-1 were horizontally transferred to p R1,and the novel fusion plasmid shared the same backbone as p R1.p D2（R5）2＿mcr-1F in the transconjugant D2（R5）2 was an Inc HI2plasmid formed upon fusion between two plasmids that originally existed in D2;the insertion of the polymyxin resistance-associated gene cassette ISApl1–mcr-1–pap2,carried by p D2＿mcr-1,into p D2＿ami led to the formation of a novel fusion plasmid,which was subsequently transferred to R5.p D6（EC）3＿mcr-1 in the transconjugant D6（EC）3 was identified to be a novel Inc FIB–Inc HI2 hybrid plasmid,which was formed upon fusion between p D6＿mcr-1 and p D6＿tet（M）in D6 through interaction with a 38-bp homologous region.The three fusion plasmids carried mcr-1 and antibiotic resistance genes.Plasmid fusion occurred at frequencies of 1.48×10^-3 to 3.94×10^-3 per recipient cell.The fusion plasmids presented high stability and could transfer their antibiotic resistance-encoding gene cassettes including mcr-1 to E.coli C600 and J53 at frequencies of 1.73×10^-3 to 2.32×10^-4 per recipient cell.Plasmid fusion events occur frequently and the mechanism was complex.Fusion plasmids can carry out stable inheritance and have strong conjugative transfer capabilities.更多还原