本文選題：彎曲菌　切入點(diǎn)：ermB基因　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：空腸彎曲菌(C. jejuni)和結(jié)腸彎曲菌(C. coli)是重要的食源性病原菌。畜禽是彎曲菌的重要宿主和天然儲(chǔ)庫(kù),抗菌藥物在畜牧生產(chǎn)中大量應(yīng)用使耐藥的彎曲菌被篩選出來,并有可能通過食物鏈傳播給人,這將給人類健康和食品安全構(gòu)成潛在威脅。耐藥基因島因同時(shí)介導(dǎo)數(shù)種甚至數(shù)類抗菌藥物的耐藥,而引起人們關(guān)注。國(guó)內(nèi)外對(duì)耐藥基因島在彎曲菌中的流行病學(xué)和遺傳背景方面的研究尚屬空白。本研究將調(diào)查我國(guó)部分地區(qū)彎曲菌的耐藥性情況,在此基礎(chǔ)上探討耐藥基因島在食品動(dòng)物源和腹瀉病人來源彎曲菌中流行及傳播機(jī)制的異同。本研究在山東、寧夏和廣東地區(qū)采集到的3083份畜禽樣本中分離到763株彎曲菌(分離率為24.7%),包括679株C. coli和84株C. jejuni。465株豬源彎曲菌中,99.1%的菌株是C. coli, C. coli依然是豬源彎曲菌的優(yōu)勢(shì)菌種；而298株禽源(雞和鴨)彎曲菌中,73.2%的菌株是C.coli, C.coli正取代C. jejuni成為我國(guó)禽源彎曲菌中的優(yōu)勢(shì)菌種。藥敏試驗(yàn)顯示C. coli對(duì)紅霉素、克林霉素和慶大霉素的耐藥率顯著高于C. jejuni。彎曲菌的多重耐藥情況嚴(yán)峻,多重耐藥率達(dá)88.3%,且C. coli的多重耐藥率(90.4%)遠(yuǎn)高于C.jejuni的多重耐藥率(71.4%)。對(duì)分離自我國(guó)多地區(qū)的1555株畜禽源及人源彎曲菌PCR檢測(cè)多藥耐藥基因ermB后獲得59株陽性菌(陽性率為3.8%；58株C. coli和1株C. jejuni),包括33株豬源、14株雞源、10株人源和2株鴨源,禽源、人源彎曲菌和C. jejuni中發(fā)現(xiàn)ermB尚屬首次。隨后對(duì)ermB的側(cè)翼結(jié)構(gòu)測(cè)定分析后發(fā)現(xiàn),28株陽性菌攜帶的多重耐藥基因島(MDRGI)可分為7個(gè)類型。其中,type Ⅲ MDRGI同時(shí)確證分離于不同年份不同地區(qū)不同宿主的16株C. coli中；在type Ⅱ MDRGI中發(fā)現(xiàn)新型磷霉素耐藥基因fosXCC。此外,PFGE和MLST分型提示ermB陽性菌在山東和上海地區(qū)發(fā)生了克隆傳播；并且攜帶MDRGIs的彎曲菌有通過食物鏈傳播給人的較大可能性。自然轉(zhuǎn)化試驗(yàn)表明7種MDRGIs及其耐藥表型均可跨彎曲菌亞種進(jìn)行傳播。另外,ermB的表達(dá)調(diào)控試驗(yàn)顯示彎曲菌DZB40和86c的ermB是誘導(dǎo)型耐藥表達(dá),其誘導(dǎo)機(jī)制與ermB的mRNA二級(jí)結(jié)構(gòu)的堿基組成相關(guān)；而27株彎曲菌的ermB則是固有型耐藥表達(dá),其表達(dá)與ermB調(diào)控區(qū)的變化有關(guān)。對(duì)山東畜禽源和上海腹瀉病人來源的110株C. coli PCR檢測(cè)耐藥基因簇aadE-sat4-aphA-3后獲得14株陽性菌(12.7%),包括7株人源、6株豬源和1株鴨源。對(duì)基因簇的側(cè)翼結(jié)構(gòu)測(cè)定分析后發(fā)現(xiàn),10株陽性菌攜帶的氨基糖苷耐藥基因島(ARGI)可分為3個(gè)類型。其中, type Ⅰ ARGI同時(shí)發(fā)現(xiàn)在分離于畜禽源和人源的C. coli中；僅發(fā)現(xiàn)在一株人源C. coli的type Ⅲ ARGI中包含全新的IS-lide元件。隨后分子分型發(fā)現(xiàn)分離自腹瀉病人的C. coli的ARGI有可能是經(jīng)食物鏈從攜帶ARGI的畜禽源C. coli中獲得。穩(wěn)定性試驗(yàn)顯示ARGIs在沒有抗生素壓力的條件下能夠穩(wěn)定存在,并介導(dǎo)傳代子與原代菌相同水平的耐藥性。耐藥基因島在64株彎曲菌中的整合位點(diǎn)分別有①：整合至cj068c與sodB之間,只發(fā)現(xiàn)在1株攜帶MDRGI的雞源C. jejuni中；②：直接插入到cj1063中,發(fā)現(xiàn)在7株人源的C. coli中,包括2種MDRGIs;③:取代cj1477c后整合至cj1476c和cadF之間,發(fā)現(xiàn)在畜禽源和人源的55株C. coli中,包括4種MDRGIs和2種ARGIs;④：直接插入到cj1500中,僅發(fā)現(xiàn)在一株攜帶ARGI的人源C. coli中。推測(cè)位點(diǎn)③是耐藥基因島在彎曲菌的染色體DNA中的整合熱點(diǎn)。綜上所述,攜帶氨基糖苷耐藥基因島的彎曲菌對(duì)臨床上用于治療彎曲菌的急性或全身性感染時(shí)使用的氨基糖苷類藥物均耐藥；攜帶多重耐藥基因島的彎曲菌對(duì)用于治療彎曲菌病的抗菌藥物(包括大環(huán)內(nèi)酯類、氟喹諾酮類、氨基糖苷類、四環(huán)素類、林可胺類)均耐藥。7種MDRGIs和3種ARGIs在我國(guó)畜禽源及人源彎曲菌中已有流行的趨勢(shì),均可跨彎曲菌亞種進(jìn)行傳播,而且攜帶MDRGIs或ARGIs的彎曲菌都有通過食物鏈傳播給人的潛在風(fēng)險(xiǎn)。另外,我國(guó)畜牧業(yè)長(zhǎng)期亞劑量的用藥,將會(huì)對(duì)攜帶誘導(dǎo)型耐藥表達(dá)ermB的彎曲菌的耐藥性誘導(dǎo)產(chǎn)生提供便利,并且促使誘導(dǎo)型耐藥的ermB進(jìn)化為固有型耐藥表達(dá)的ermB。
[Abstract]:Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) is an important food borne pathogen. Livestock and poultry is an important host of Campylobacter and natural antibacterial drug reservoir, Campylobacter widely used in livestock production in the resistant were screened and may spread to people through the food chain, which will a potential threat to human health and food safety. At the same time because of resistance Island resistance gene mediates a variety of even a few antibiotics, and attracted attention. The domestic and foreign research on epidemiology and genetic background in Campylobacter in resistance gene island is still blank. This study will investigate the situation of drug resistance of Campylobacter in parts of China in China, based on the similarities and differences of resistance gene island in the animal food source and source of Campylobacter in patients with diarrhea prevalence and transmission mechanism. The research in Shandong, 3083 livestock and poultry to sample collected from Ningxia and Guangdong area Isolated 763 strains of Campylobacter in (the isolation rate was 24.7%), including 679 strains of C. coli and C. jejuni.465 84 strains from swine Campylobacter, 99.1% strains are C. coli, C. coli is still the dominant strain of swine Campylobacter; and 298 strains of avian (chicken and duck) in Campylobacter 73.2%, the strain is C.coli C.coli, replacing C. jejuni as the dominant strain of avian Campylobacter in China. Drug sensitivity test showed that C. coli of erythromycin, clindamycin and gentamicin multi drug resistance rate was significantly higher than that of C. jejuni. of Campylobacter grim, multi drug resistance rate was 88.3%, and the multi drug resistant C. coli the rate of multi drug resistance (90.4%) is much higher than the rate of C.jejuni (71.4%). Of 1555 strains of livestock and poultry sources and human Campylobacter PCR detection region isolated from the multidrug resistance gene ermB obtained 59 positive bacteria (positive rate was 3.8%; 58 strains and 1 strains of C. coli C. jejuni), including 33 strains swine, 14 From chicken, 10 strains and 2 strains of duck origin source, avian, human Campylobacter and C. found in jejuni ermB for the first time. Then the flank structure determination of ermB analysis showed that 28 strains of Gram-positive bacteria carrying multidrug resistance gene Island (MDRGI) can be divided into 7 types. Among them, type MDRGI also confirmed the separation of III in different regions of different year and host of the 16 strains of C. coli; hormone resistant gene fosXCC. model fosfomycin also found in type II MDRGI, PFGE and MLST type ermB positive bacteria in Shandong and Shanghai area has undergone clonal propagation; Campylobacter and MDRGIs are more likely to carry through the food chain spread to people. The natural transformation test showed that 7 kinds of MDRGIs and its drug-resistant phenotype can be spread across Campylobacter subspecies. In addition, expression regulation test of ermB display DZB40 and 86C of Campylobacter ermB is inducible resistance expression, its mechanism of induction and ermB MRNA two level structure of base composition; and 27 strains of Campylobacter ermB is the expression of innate resistance, its expression is related with the change of ermB regulatory region of aadE-sat4-aphA-3. 110 strains of C. coli PCR detection of drug resistance gene cluster of livestock and poultry in Shandong and Shanghai from the source of patients with diarrhea after 14 strains of positive bacteria (12.7%). Including 7 strains of human source, 6 strains of swine and 1 strains of duck origin. Determination and analysis of flanking structure of the gene cluster found 10 positive bacteria carrying the aminoglycoside resistance gene Island (ARGI) can be divided into 3 types. Among them, type I ARGI also found in isolated from livestock and poultry sources and human. C. coli; found only contains IS-lide element in a new strain of human C. coli type 3 ARGI. Then the molecular type was isolated from diarrhea patients C. coli ARGI may be through the food chain from animal source C. carrying ARGI in coli. The stability test showed that ARGIs in the There are no antibiotics can stabilize the pressure condition, and drug resistance mediated by primary and passage sub level. The same bacteria resistance gene Island integration sites in 64 strains of Campylobacter in respectively: integration to cj068c and sodB, only to find the chicken C. jejuni carrying MDRGI in strain 1; II: insert into cj1063, found in 7 strains of human origin C. coli, including 2 MDRGIs; 3: substitution of cj1477c to cj1476c and cadF integration between the 55 strains of C. coli, found in animal sources and human sources, including 4 MDRGIs and 2 ARGIs; 4: direct insertion into the cj1500. Only found human C. coli carrying ARGI in a line. That is the integration of the site of chromosome DNA hot resistance gene island in Campylobacter in summary, Campylobacter with aminoglycoside resistant gene island of clinically used in the treatment of acute Campylobacter or systemic infection with ammonia Glucoside type of drug resistance; multidrug resistance gene carrying Campylobacter island for antimicrobial treatment of Campylobacter diseases (including macrolide and fluoroquinolones, aminoglycosides, tetracyclines, lincosamide) were resistant.7 MDRGIs and 3 ARGIs existing in livestock and poultry sources in China and the source of bending from the popularity of the trend, can cross the spread of Campylobacter subspecies, and Campylobacter carrying MDRGIs or ARGIs have the potential risk of food chain spread to people. In addition, long-term mild dose of animal husbandry industry in China, will carry on inducible resistance resistance of Campylobacter table up to ermB induced facilitation ermB, and prompted the evolution of inducible resistance for the expression of innate resistance ermB.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.61
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本文編號(hào)：1604518