【摘要】：空腸彎曲菌(C.jejuni)和結(jié)腸彎曲菌(C.coli)是人源及動物源中傳播范圍較廣、致病性較強的食源性病原菌。大環(huán)內(nèi)酯類藥物是治療彎曲菌感染的首選抗菌藥物之一。近年來,由于抗菌藥物的大量使用,彎曲菌對大環(huán)內(nèi)酯類藥物的耐藥率呈明顯上升趨勢,而介導大環(huán)內(nèi)酯類耐藥的核糖體甲基化酶基因ermB在彎曲菌中的出現(xiàn),給有效控制和治療耐藥彎曲菌帶來很大挑戰(zhàn)。另外,ermB基因穩(wěn)定的水平傳播特性也可能會導致耐紅霉素彎曲菌的大范圍流行。因此,本研究旨在調(diào)查ermB基因近年來在中國地區(qū)的流行及傳播特征,并在此基礎上進一步研究攜帶ermB基因彎曲菌的適應性變化特點和相關(guān)機制。本研究于2015年在山東、上海、廣東地區(qū)采集到的1134份豬源(n=544)、雞源(n=590)糞便或盲腸內(nèi)容物樣本中共分離得到370株彎曲菌(分離率:32.6%),運用PCR對進行ermB基因檢測共得到37株ermB陽性C.coli,陽性率為10.0%。結(jié)合2013與2014年的數(shù)據(jù)分析,2013～2015年間ermB基因的陽性分離率分別為2.1%、8.2%、10.0%。而山東、上海、廣東三個地區(qū)在三年間的總陽性率分別為1.1%、3.3%、15.1%。廣東地區(qū)流行率較高,且呈現(xiàn)明顯上升特點。上海及廣東地區(qū)的ermB陽性菌株表現(xiàn)出較高的同源性,并出現(xiàn)了與2011年分離自人源ermB陽性菌高度相似的譜型,推測優(yōu)勢克隆型己在動物源與人源之間進行跨地域、跨時間的克隆傳播。鑒于ermB陽性彎曲菌在我國部分地區(qū)的流行率呈上升的特征,推測ermB基因可能導致彎曲菌在適應性方面產(chǎn)生變化。為了解其適應性變化特點,本研究構(gòu)建了兩對遺傳背景一致的ermB耐藥工程菌株,并進行體內(nèi)、體外試驗。體內(nèi)、體外生長試驗結(jié)果均表明,攜帶ermB的菌株生長、定殖能力上較敏感菌相比均出現(xiàn)下降;而在體內(nèi)競爭試驗中,ermB菌株在接種初期表現(xiàn)出適應性代價,而在接種后期發(fā)生適應性回復現(xiàn)象。代償菌株經(jīng)體內(nèi)體外生長、競爭的驗證試驗后,確證其出現(xiàn)了一定的適應性回復。為探索這一不同的適應性變化機制,嘗試運用定量蛋白質(zhì)組學方法研究發(fā)生適應性改變時彎曲菌在蛋白水平上的差異變化。運用同重同位素標簽標記定量法(iTRAQ)進行篩選,耐藥菌相對于敏感菌有123個差異顯著蛋白,而代償菌相對于耐藥菌有25個差異顯著蛋白。同時還運用了雙向熒光差異凝膠電泳法(2D-DIGE)篩選,在代償菌中發(fā)現(xiàn)13個與耐藥菌相比差異表達的蛋白。通過GO、Pathway、COG等分析,這些差異蛋白主要影響了細菌的能量代謝及生長定殖能力。將上述結(jié)果與相關(guān)文獻報道整合分析后得出,ermB陽性彎曲菌在體內(nèi)競爭初期發(fā)生的適應性代價主要是由于運動能力下降及能量代謝失衡導致,而ErmB及鞭毛蛋白的高表達是影響能量平衡的關(guān)鍵因素。另外,研究還發(fā)現(xiàn)ermB陽性彎曲菌可通過自身的調(diào)節(jié)恢復能量的攝取和利用水平,同時與細菌定殖、運動相關(guān)蛋白的二次代償可增強細菌繁殖能力,從而回復適應性。綜上所述,ermB陽性彎曲菌在我國部分地區(qū)的流行呈上升特征,且有可能已在人畜間發(fā)生相互傳播,其原因除了抗菌藥物的篩選壓力外,還可能與攜帶ermB基因菌株存在適應性回復的現(xiàn)象有關(guān)。上述現(xiàn)象可能會使彎曲菌在獲得ermB基因后帶來更大范圍的流行傳播。本研究結(jié)果加深了我們對耐藥彎曲菌在流行傳播上的認識,同時為尋找控制耐藥彎曲菌傳播的方法提供了理論基礎。
[Abstract]:Campylobacter jejuni (C. jeejunii) and Campylobacter coli (C. coli) are the source of human origin and the source of animal. Macrolides are one of the most preferred antimicrobial agents for the treatment of Campylobacter infections. In recent years, due to the extensive use of the antibacterial drugs, the resistance rate of the curved bacteria to the macrolides is obviously increased, and the occurrence of the ribosomal methylase gene erB-mediated macrolide-resistant ribosomal methylase gene erB in the bending bacteria is greatly challenged for the effective control and treatment of the drug-resistant bending bacteria. In addition, the stable level-propagation characteristics of the mB gene may also lead to a wide spread of resistance to erythromycin-bending bacteria. Therefore, the purpose of this study is to investigate the prevalence and transmission characteristics of the ermB gene in China. A total of 370 strains of Campylobacter (isolation rate: 32.6%) were isolated from 1134 pig sources (n = 544), chicken source (n = 590), or cecum contents collected in Shandong, Shanghai and Guangdong Province in 2015. The positive rate was 10.0%. According to the data analysis of 2013 and 2014, the positive separation rate of the mB gene in 2013-2015 was 2.1%, 80.2% and 10.0%, respectively. The total positive rates of the three regions in Shandong, Shanghai and Guangdong were 1. 1%, 3. 3% and 15. 1%, respectively. in Guangdong, that prevalence rate of the Guangdong area is high, and the characteristic of the obvious increase is present. The mB-positive strains in Shanghai and Guangdong showed high homology, and showed a highly similar profile with that of the human source, which was isolated from human sources in 2011, and it was speculated that the dominant clone had a cross-region and cross-time clone propagation between the source of the animal and the human source. In view of the increasing prevalence of the mB-positive Campylobacter in some parts of China, it is suggested that the mB gene may lead to a change in the adaptability of the Campylobacter. In order to understand the characteristics of its adaptability, two pairs of metB-resistant engineering strains with the same genetic background were constructed and tested in vivo and in vitro. In vivo and in vitro, the results of in vitro growth test showed that the growth and colonization ability of the strain carrying the mB showed a decrease compared with that of the sensitive bacteria. In the in-vivo competition test, the mB strain showed the adaptive cost in the early stage of the inoculation, and the adaptive response at the later stage of the inoculation. After the test of the in vitro growth and competition of the compensatory strain, a certain adaptive response was confirmed. In order to explore this different mechanism of adaptive change, the difference in protein level of Campylobacter in the case of adaptive change was studied by means of quantitative proteomics. It was found that the drug-resistant bacteria have 123 distinct proteins with respect to the sensitive bacteria, and 25 of the compensation bacteria have a significant difference with the drug-resistant bacteria. At the same time, two-way fluorescence differential gel electrophoresis (2D-DIGE) was used to screen, and 13 proteins that were differentially expressed in comparison with drug-resistant bacteria were found in the compensatory bacteria. Through the analysis of GO, Pathways, COG and the like, these differential proteins mainly affect the energy metabolism and the growth and colonization ability of the bacteria. It is concluded that the adaptive cost of the ermB positive bending bacteria in the early stage of the in vivo competition is mainly due to the decrease of the exercise capacity and the imbalance of energy metabolism, while the high expression of ErmB and flagellin is the key factor affecting the energy balance. In addition, the study also found that the mB-positive Campylobacter can restore the uptake and utilization level of the energy through its own regulation, and at the same time colonize with the bacteria, the secondary compensation of the motion-related protein can enhance the bacterial reproduction ability, so as to respond to the adaptability. In conclusion, the epidemic of the mB-positive Campylobacter in some parts of our country is on the rise, and it is possible to spread among the human animals. The reason of this is that, in addition to the screening pressure of the antibacterial drugs, it is also related to the phenomenon of the adaptive response of the MB gene. The above phenomenon may lead to a more widespread spread of the Campylobacter after obtaining the mB gene. The results of this study have deepened our understanding of drug-resistant bending bacteria in the epidemic, and also provides a theoretical basis for finding a method to control the propagation of drug-resistant curved bacteria.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.61

【參考文獻】

相關(guān)期刊論文 前10條

1 ;“基于濾膜通透性彎曲菌新檢測方法及彎曲菌超級耐藥島篩查技術(shù)”培訓班會議紀要[J];疾病監(jiān)測;2016年05期

2 張鑫;佘丹陽;劉又寧;王睿;邸秀珍;梁蓓蓓;王悅;;細菌定量蛋白質(zhì)組學研究英文文獻計量分析[J];中華內(nèi)科雜志;2014年07期

3 鞏紅飛;柳軍璽;邸多隆;;蛋白質(zhì)組學研究中的分離分析技術(shù)及其研究進展[J];氨基酸和生物資源;2014年02期

4 蔣月;盛鵬飛;張陸;;空腸彎曲桿菌對氟喹諾酮類和大環(huán)內(nèi)酯類藥物耐藥機制研究進展[J];中國人獸共患病學報;2013年09期

5 錢小紅;;定量蛋白質(zhì)組學分析方法[J];色譜;2013年08期

6 周愿;單亦初;張麗華;張玉奎;;基于穩(wěn)定同位素標記的蛋白質(zhì)組學定量方法研究進展[J];色譜;2013年06期

7 杜俊變;王麗惠;段江燕;;2D-DIGE技術(shù)在蛋白質(zhì)組學中的應用[J];生物學雜志;2011年03期

8 楊國堂;李艷艷;張偉;孟令羽;;蛋白質(zhì)組學及其技術(shù)體系[J];食品與藥品;2010年05期

9 朱金蕾;張鍇;何錫文;張玉奎;;基于質(zhì)譜技術(shù)蛋白質(zhì)定量方法的研究進展[J];分析化學;2010年03期

10 王華丙;張振義;包銳;陳宇星;;ABC轉(zhuǎn)運蛋白的結(jié)構(gòu)與轉(zhuǎn)運機制[J];生命的化學;2007年03期

相關(guān)博士學位論文 前3條

1 鄧鳳如;耐藥基因島在我國部分地區(qū)動物源和人源彎曲菌中的流行和傳播[D];中國農(nóng)業(yè)大學;2016年

2 李會;空腸彎曲菌酰胺醇類誘導耐藥株與敏感株的組學比較研究[D];中國農(nóng)業(yè)大學;2015年

3 馬立才;典型肉雞生產(chǎn)鏈中彎曲菌耐藥性調(diào)查及風險評估研究[D];中國農(nóng)業(yè)大學;2014年

，

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