本文選題：禽大腸埃希菌　切入點(diǎn)：blaCTX-M　出處：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：超廣譜β-內(nèi)酰胺酶(extented spectrumβ-lactamases,ESBLs)是細(xì)菌對(duì)三代頭孢耐藥的主要機(jī)制,大腸埃希菌是ESBLs最主要的產(chǎn)生菌。ESBLs基因型有多種,近十年來,CTX-M型已逐漸代替TEM型和SHV型而成為散播最為廣泛的基因型。本試驗(yàn)旨在通過檢測(cè)不同來源雞源大腸埃希菌(分離自市售健康雞肉和疑似大腸埃希菌病死亡的病死雞肝臟)中CTX-M型ESBLs的分子流行特征基礎(chǔ)上,分析不同來源、不同亞群的blaCTX-M散播機(jī)制及遺傳背景,以期為臨床控制blaCTX-M的散播及防治產(chǎn)CTX-M型雞大腸埃希菌感染提供理論基礎(chǔ)。用微量肉湯稀釋法測(cè)定106株雞大腸埃希菌(包括18株雞肉分離菌和88株病料分離菌)對(duì)氨芐西林、頭孢噻呋、頭孢噻肟、慶大霉素、阿米卡星、多西環(huán)素、氟苯尼考和恩諾沙星8種抗菌藥物的最小抑菌濃度(MIC),結(jié)果顯示不同來源分離菌對(duì)藥物的耐藥特征相似,耐藥率均在18.2~94.4%。76株對(duì)三代頭孢耐藥的雞大腸埃希菌(包括10株雞肉分離菌和66株病料分離菌)的ESBLs檢測(cè)結(jié)果表明:10株雞肉分離菌中有5株菌攜帶blaCTX-M,檢出率為50%,其中4株為CTX-M-9亞群(3株blaCTX-M-14和1株blaCTX-M-65),1株為CTX-M-1亞群(1株blaCTX-M-55)。66株病料分離菌中有63株菌攜帶blaCTX-M,檢出率高達(dá)95.5%,其中屬于CTX-M-9亞群有25株(21株blaCTX-M-65和4株blaCTX-M-14)、CTX-M-1亞群有23株(22株blaCTX-M-55和1株bla CTX-M-15)、兩種不同亞群雜合的有7株(4株blaCTX-M-64和3株bla CTX-M-123),此外尚有8株分離菌未確定具體的基因亞型。說明目前河南省雞大腸埃希菌中攜帶的CTX-M基因主要屬于CTX-M-9亞群和CTX-M-1亞群。采用PCR定位技術(shù)檢測(cè)不同亞群的blaCTX-M上、下游基因序列。結(jié)果顯示:不同亞群的blaCTX-M上游均含有ISEcp1,但下游插入序列不同,其中CTX-M-1亞群、blaCTX-M-64和blaCTX-M-123的下游為ORF477,CTX-M-9的下游為IS903。此外,結(jié)果表明在CTX-M-9亞群中有高達(dá)80.5%的ISEcp1被其他插入序列截?cái)?而CTX-M-1亞群中,僅有12.5%的ISEcp1被IS26所截?cái)?說明CTX-M-9亞群的上、下游插入序列更易被IS26、IS10和其他未知插入序列截?cái)?且下游插入序列結(jié)構(gòu)更復(fù)雜,其末端經(jīng)常有1個(gè)18bp的反向重復(fù)序列(IRL)。質(zhì)粒不相容群結(jié)果顯示本批受試菌共檢出13種質(zhì)粒復(fù)制子類型,其中有67株菌(91.8%,67/73)的質(zhì)粒復(fù)制子類型不低于3種。不同blaCTX-M亞群的菌株攜帶質(zhì)粒復(fù)制子類型基本一致,且雞肉分離菌和病料分離菌攜帶的質(zhì)粒復(fù)制子無明顯差異。均為IncFII、IncK和IncFIB的檢出率最高,達(dá)82%~86%,均未檢出IncX、IncL/M、IncW、IncY和Inc FrepB復(fù)制子。說明本批受試菌多數(shù)均攜帶有多個(gè)質(zhì)粒復(fù)制子,有助于提高質(zhì)粒的宿主適應(yīng)性和耐藥基因的散播。結(jié)果顯示13株原保存菌與55株病料分離菌攜帶的復(fù)制子差異較明顯,其中IncFIA、IncA/C和IncT僅在病料分離菌中檢出,而IncI1和IncB在前者中的檢測(cè)率明顯高于后者。質(zhì)粒接合試驗(yàn)結(jié)果表明,63.0%(46/73)的產(chǎn)blaCTX-M菌株可以成功將耐三代頭孢菌素的耐藥質(zhì)粒傳遞給受體菌J53,其中5株雞肉分離菌的質(zhì)粒均全部發(fā)生了傳遞,13株原保存菌中有9株菌質(zhì)粒發(fā)生了接合,接合率為69.2%;55株病料分離菌中有32株菌質(zhì)粒發(fā)生了接合,接合率為58.2%,三者之間無明顯差異。說明接合性質(zhì)粒在CTX-M散播中發(fā)揮了重要的作用。種系發(fā)育分型的結(jié)果顯示,73株產(chǎn)blaCTX-M的大腸埃希菌中,種系發(fā)育群A、B1、B2和D類的菌株比例分別為31.5%、46.6%、4.1%和17.8%。20株不產(chǎn)ESBLs的敏感大腸埃希菌屬于種系發(fā)育群A、B1、B2和D類的菌株比例分別為30%、65%、0%和5%。其中在產(chǎn)CTX-M受試菌中,有21.9%菌株具有致病力,而不產(chǎn)ESBLs的敏感菌中僅有5%具有致病力,且高致病力菌株僅出現(xiàn)在產(chǎn)CTX-M菌中,說明產(chǎn)CTX-M雞大腸埃希菌的致病力可能較不產(chǎn)酶的細(xì)菌強(qiáng),但尚須進(jìn)一步試驗(yàn)證實(shí)。MLST結(jié)果顯示,73株產(chǎn)blaCTX-M大腸埃希菌中共檢出26個(gè)ST型,20株不產(chǎn)ESBLs菌株共檢出10個(gè)ST型,其中ST155和ST359在blaCTX-M菌株和不產(chǎn)ESBLs中均有檢出,且ST155在兩類細(xì)菌中的檢出率均最高。本批受試菌中共檢出10個(gè)新發(fā)現(xiàn)的ST型,其中ST4753中的fumC612和ST4752中的mdh382為發(fā)現(xiàn)的新的等位基因,相關(guān)序列均已提交至MLST數(shù)據(jù)庫。不僅說明本批受試菌的親緣關(guān)系復(fù)雜,分屬于不同的克隆株,且變異速度快,也說明克隆在CTX-M的散播中僅發(fā)揮了次要的作用。綜上所述,目前河南省產(chǎn)CTX-M雞大腸埃希菌中流行的基因亞型以CTX-M-1亞群和CTX-M-9亞群為主,有少量的雜合基因檢出,CTX-M基因亞型日趨復(fù)雜多樣。產(chǎn)CTX-M菌株的親緣關(guān)系復(fù)雜,來源也呈現(xiàn)多樣化;ISEcp1插入序列和接合性質(zhì)粒在blaCTX-M基因散播過程中發(fā)揮了至關(guān)重要的作用,而克隆僅發(fā)揮了次要的作用。
[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.

【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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