禽大肠埃希菌不同CTX-M亚群的散播机制及遗传特性

发布时间：2018-04-09 23:02

本文选题：禽大肠埃希菌　切入点：blaCTX-M　出处：《河南农业大学》2015年硕士论文

【摘要】：超广谱β-内酰胺酶(extented spectrumβ-lactamases,ESBLs)是细菌对三代头孢耐药的主要机制,大肠埃希菌是ESBLs最主要的产生菌。ESBLs基因型有多种,近十年来,CTX-M型已逐渐代替TEM型和SHV型而成为散播最为广泛的基因型。本试验旨在通过检测不同来源鸡源大肠埃希菌(分离自市售健康鸡肉和疑似大肠埃希菌病死亡的病死鸡肝脏)中CTX-M型ESBLs的分子流行特征基础上,分析不同来源、不同亚群的blaCTX-M散播机制及遗传背景,以期为临床控制blaCTX-M的散播及防治产CTX-M型鸡大肠埃希菌感染提供理论基础。用微量肉汤稀释法测定106株鸡大肠埃希菌(包括18株鸡肉分离菌和88株病料分离菌)对氨苄西林、头孢噻呋、头孢噻肟、庆大霉素、阿米卡星、多西环素、氟苯尼考和恩诺沙星8种抗菌药物的最小抑菌浓度(MIC),结果显示不同来源分离菌对药物的耐药特征相似,耐药率均在18.2~94.4%。76株对三代头孢耐药的鸡大肠埃希菌(包括10株鸡肉分离菌和66株病料分离菌)的ESBLs检测结果表明:10株鸡肉分离菌中有5株菌携带blaCTX-M,检出率为50%,其中4株为CTX-M-9亚群(3株blaCTX-M-14和1株blaCTX-M-65),1株为CTX-M-1亚群(1株blaCTX-M-55)。66株病料分离菌中有63株菌携带blaCTX-M,检出率高达95.5%,其中属于CTX-M-9亚群有25株(21株blaCTX-M-65和4株blaCTX-M-14)、CTX-M-1亚群有23株(22株blaCTX-M-55和1株bla CTX-M-15)、两种不同亚群杂合的有7株(4株blaCTX-M-64和3株bla CTX-M-123),此外尚有8株分离菌未确定具体的基因亚型。说明目前河南省鸡大肠埃希菌中携带的CTX-M基因主要属于CTX-M-9亚群和CTX-M-1亚群。采用PCR定位技术检测不同亚群的blaCTX-M上、下游基因序列。结果显示:不同亚群的blaCTX-M上游均含有ISEcp1,但下游插入序列不同,其中CTX-M-1亚群、blaCTX-M-64和blaCTX-M-123的下游为ORF477,CTX-M-9的下游为IS903。此外,结果表明在CTX-M-9亚群中有高达80.5%的ISEcp1被其他插入序列截断,而CTX-M-1亚群中,仅有12.5%的ISEcp1被IS26所截断;说明CTX-M-9亚群的上、下游插入序列更易被IS26、IS10和其他未知插入序列截断,且下游插入序列结构更复杂,其末端经常有1个18bp的反向重复序列(IRL)。质粒不相容群结果显示本批受试菌共检出13种质粒复制子类型,其中有67株菌(91.8%,67/73)的质粒复制子类型不低于3种。不同blaCTX-M亚群的菌株携带质粒复制子类型基本一致,且鸡肉分离菌和病料分离菌携带的质粒复制子无明显差异。均为IncFII、IncK和IncFIB的检出率最高,达82%~86%,均未检出IncX、IncL/M、IncW、IncY和Inc FrepB复制子。说明本批受试菌多数均携带有多个质粒复制子,有助于提高质粒的宿主适应性和耐药基因的散播。结果显示13株原保存菌与55株病料分离菌携带的复制子差异较明显,其中IncFIA、IncA/C和IncT仅在病料分离菌中检出,而IncI1和IncB在前者中的检测率明显高于后者。质粒接合试验结果表明,63.0%(46/73)的产blaCTX-M菌株可以成功将耐三代头孢菌素的耐药质粒传递给受体菌J53,其中5株鸡肉分离菌的质粒均全部发生了传递,13株原保存菌中有9株菌质粒发生了接合,接合率为69.2%;55株病料分离菌中有32株菌质粒发生了接合,接合率为58.2%,三者之间无明显差异。说明接合性质粒在CTX-M散播中发挥了重要的作用。种系发育分型的结果显示,73株产blaCTX-M的大肠埃希菌中,种系发育群A、B1、B2和D类的菌株比例分别为31.5%、46.6%、4.1%和17.8%。20株不产ESBLs的敏感大肠埃希菌属于种系发育群A、B1、B2和D类的菌株比例分别为30%、65%、0%和5%。其中在产CTX-M受试菌中,有21.9%菌株具有致病力,而不产ESBLs的敏感菌中仅有5%具有致病力,且高致病力菌株仅出现在产CTX-M菌中,说明产CTX-M鸡大肠埃希菌的致病力可能较不产酶的细菌强,但尚须进一步试验证实。MLST结果显示,73株产blaCTX-M大肠埃希菌中共检出26个ST型,20株不产ESBLs菌株共检出10个ST型,其中ST155和ST359在blaCTX-M菌株和不产ESBLs中均有检出,且ST155在两类细菌中的检出率均最高。本批受试菌中共检出10个新发现的ST型,其中ST4753中的fumC612和ST4752中的mdh382为发现的新的等位基因,相关序列均已提交至MLST数据库。不仅说明本批受试菌的亲缘关系复杂,分属于不同的克隆株,且变异速度快,也说明克隆在CTX-M的散播中仅发挥了次要的作用。综上所述,目前河南省产CTX-M鸡大肠埃希菌中流行的基因亚型以CTX-M-1亚群和CTX-M-9亚群为主,有少量的杂合基因检出,CTX-M基因亚型日趋复杂多样。产CTX-M菌株的亲缘关系复杂,来源也呈现多样化;ISEcp1插入序列和接合性质粒在blaCTX-M基因散播过程中发挥了至关重要的作用,而克隆仅发挥了次要的作用。
[Abstract]:ESBLs (extented spectrum beta -lactamases, ESBLs) is the main mechanism of the bacterial resistance to the three generation cephalosporins, Escherichia coli is a variety of ESBLs main producing strains of genotype.ESBLs, over the past ten years, CTX-M has been gradually replaced by TEM and SHV and become the most spread of genotype widely. This test is designed to detect different sources of avian Escherichia coli (isolated from commercially available chicken Health and suspected death of Escherichia coli disease of dead chicken liver) molecular epidemiological characteristics of CTX-M type ESBLs based on the analysis of different sources, different subsets of blaCTX-M spread mechanism and genetic background, in order to provide the theory of the foundation for the clinical prevention and control the spread of blaCTX-M producing CTX-M type avian Escherichia coli infection. 106 strains of avian Escherichia coli were determined by broth microdilution method (including 18 strains of chicken isolates and 88 strains of bacteria isolated from diseased) of ampicillin, Ceftiofur, cefotaxime, gentamicin, Amikacin, doxycycline, the minimum inhibitory concentration of florfenicol and enrofloxacin 8 antimicrobial agents (MIC), results show that the characteristics of drug resistance of bacteria isolated from different sources are similar, the resistance rate of 18.2~94.4%.76 strain of the three generation cephalosporin resistant Escherichia coli (chicken including 10 strains of chicken isolates and 66 strains of bacteria isolated from diseased) ESBLs detection results showed that 10 strains of bacteria isolated from chicken in 5 strains carrying blaCTX-M, the detection rate was 50%, including 4 strains of subgroup CTX-M-9 (3 strains and 1 strains of blaCTX-M-14 blaCTX-M-65), 1 strains (1 strains of subgroup CTX-M-1 blaCTX-M-55).66 plant disease material isolated bacteria in 63 strains carrying blaCTX-M, the detection rate of 95.5%, which belongs to the CTX-M-9 subgroup of 25 strains (21 strains and 4 strains of blaCTX-M-65 blaCTX-M-14, CTX-M-1) subgroup of 23 strains (22 strains and 1 strains of blaCTX-M-55 bla CTX-M-15), two different subsets of heterozygous there were 7 strains (4 Strains blaCTX-M-64 and 3 strains of BLA CTX-M-123), there are also 8 strains did not determine the specific subtype. The carrying CTX-M gene of avian Escherichia coli in Henan province mainly belong to the CTX-M-9 subgroup and CTX-M-1 subgroup. BlaCTX-M detection of different subsets of the PCR positioning technology, the downstream gene sequence. The results showed blaCTX-M: upstream of different subsets contain ISEcp1, but inserted into the downstream sequences, including CTX-M-1 subsets, blaCTX-M-64 and blaCTX-M-123 downstream ORF477, downstream of CTX-M-9 IS903. in addition, the results indicated that in CTX-M-9 subsets in up to 80.5% of ISEcp1 by other insertion sequence truncation, and subgroup CTX-M-1, only 12.5% the ISEcp1 IS26 CTX-M-9 subgroup truncation; on the downstream insertion sequence is more likely to be IS26, IS10 and other unknown insertion sequence truncation, and inserted into the downstream sequence structure is more complex, the end often has 1 18B The inverted repeat sequence of P (IRL). The incompatible group showed the number of tested bacteria were detected in 13 kinds of plasmid replicon types, of which 67 strains (91.8%, 67/73) plasmid replicons of not less than 3. Different types of subgroup blaCTX-M strains carrying plasmid replicon type is almost identical. And the chicken isolates and isolates the disease material carrying plasmid replicon. No significant differences were IncFII, IncK and IncFIB was the highest, up to 82%~86%, were not detected in IncX, IncL/M, IncW, IncY and Inc FrepB replicon. The number of tested strains most carry multiple plasmid replication son, help to improve the spread of host adaptability and resistance gene plasmid. The results showed that 13 strains of bacteria and 55 strains of primary disease material separation replicon strain carrying obviousdifferences, including IncFIA, IncA/C and IncT were detected only in material isolated from disease, while IncI1 and IncB detection in the former was significantly high In the latter. Plasmid conjugation test results showed that 63% (46/73) of the blaCTX-M producing strains can transfer plasmid successfully to the three generation cephalosporin resistant bacteria J53 receptor, the plasmids of 5 strains of bacteria were isolated chicken all occurred in passing, 13 strains of bacteria in raw 9 strains had plasmid bonding, bonding the rate is 69.2%; 55 strains of bacteria isolated from disease material in 32 strains of bacteria had plasmid bonding, bonding rate was 58.2%, no significant difference between the three. That conjugative plasmid plays an important role in the spread of CTX-M. Phylogenetic classification results showed that 73 strains of blaCTX-M producing Escherichia coli phylogenetic group, A, B1, B2 and D strain ratio were 31.5%, 46.6%, and 4.1% 17.8%.20 strains were sensitive to Escherichia coli producing ESBLs belong to phylogenetic group A, B1, B2 and D strain ratio were 30%, 65%, 0% and 5%. in production by CTX-M test bacteria, 21.9% strains With pathogenicity, while only 5% sensitive bacteria producing ESBLs has no pathogenicity, and highly pathogenic strains appear only in CTX-M producing strains, indicating CTX-M producing Escherichia coli virulence may be less enzyme producing bacteria, but still need to be further confirmed that the.MLST test results showed that 73 strains of blaCTX-M Escherichia coli were detected in the 26 ST type, 20 strains of ESBLs producing strains were detected in 10 ST, ST155 and ST359 in blaCTX-M strains and production were detected in ESBLs and ST155 in two kinds of bacteria detection rate was the highest. The number of tested bacteria were detected in the 10 newly discovered ST the ST4753 type, fumC612 and ST4752 in mdh382 for the discovery of new alleles were related sequence has been submitted to the MLST database. The number of tested strains not only shows that the relationship is complex, belong to different clones, and the variation of speed, also shows that cloning in the spread of CTX-M in Only play a minor role. In conclusion, popular in Henan province CTX-M producing Escherichia coli in the genotype CTX-M-1 and CTX-M-9 subgroup, there is a small amount of heterozygous gene detection, CTX-M genotype is more complex and diverse. The phylogenetic relationship of the strains producing CTX-M complex, diversified sources; ISEcp1 the insertion sequence and conjugative plasmid spread in blaCTX-M gene plays a crucial role in the process of cloning, only play a minor role.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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