攜帶ermS基因畜禽源彎曲菌的流行及適應(yīng)性機(jī)制的研究
發(fā)布時(shí)間：2018-11-19 22:31
【摘要】：空腸彎曲菌(C.jejuni)和結(jié)腸彎曲菌(C.coli)是人源及動(dòng)物源中傳播范圍較廣、致病性較強(qiáng)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(lái),由于抗菌藥物的大量使用,彎曲菌對(duì)大環(huán)內(nèi)酯類藥物的耐藥率呈明顯上升趨勢(shì),而介導(dǎo)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(lái)很大挑戰(zhàn)。另外,ermB基因穩(wěn)定的水平傳播特性也可能會(huì)導(dǎo)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(lái)在中國(guó)地區(qū)的流行及傳播特征,并在此基礎(chǔ)上進(jìn)一步研究攜帶ermB基因彎曲菌的適應(yīng)性變化特點(diǎn)和相關(guān)機(jī)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(yùn)用PCR對(duì)進(jìn)行ermB基因檢測(cè)共得到37株ermB陽(yáng)性C.coli,陽(yáng)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(yáng)性分離率分別為2.1%、8.2%、10.0%。而山東、上海、廣東三個(gè)地區(qū)在三年間的總陽(yáng)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(diǎn)。上海及廣東地區(qū)的ermB陽(yáng)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(yáng)性菌高度相似的譜型,推測(cè)優(yōu)勢(shì)克隆型己在動(dòng)物源與人源之間進(jìn)行跨地域、跨時(shí)間的克隆傳播。鑒于ermB陽(yáng)性彎曲菌在我國(guó)部分地區(qū)的流行率呈上升的特征,推測(cè)ermB基因可能導(dǎo)致彎曲菌在適應(yīng)性方面產(chǎn)生變化。為了解其適應(yīng)性變化特點(diǎn),本研究構(gòu)建了兩對(duì)遺傳背景一致的ermB耐藥工程菌株,并進(jìn)行體內(nèi)、體外試驗(yàn)。體內(nèi)、體外生長(zhǎng)試驗(yàn)結(jié)果均表明,攜帶ermB的菌株生長(zhǎng)、定殖能力上較敏感菌相比均出現(xiàn)下降;而在體內(nèi)競(jìng)爭(zhēng)試驗(yàn)中,ermB菌株在接種初期表現(xiàn)出適應(yīng)性代價(jià),而在接種后期發(fā)生適應(yīng)性回復(fù)現(xiàn)象。代償菌株經(jīng)體內(nèi)體外生長(zhǎng)、競(jìng)爭(zhēng)的驗(yàn)證試驗(yàn)后,確證其出現(xiàn)了一定的適應(yīng)性回復(fù)。為探索這一不同的適應(yīng)性變化機(jī)制,嘗試運(yùn)用定量蛋白質(zhì)組學(xué)方法研究發(fā)生適應(yīng)性改變時(shí)彎曲菌在蛋白水平上的差異變化。運(yùn)用同重同位素標(biāo)簽標(biāo)記定量法(iTRAQ)進(jìn)行篩選,耐藥菌相對(duì)于敏感菌有123個(gè)差異顯著蛋白,而代償菌相對(duì)于耐藥菌有25個(gè)差異顯著蛋白。同時(shí)還運(yùn)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(gè)與耐藥菌相比差異表達(dá)的蛋白。通過GO、Pathway、COG等分析,這些差異蛋白主要影響了細(xì)菌的能量代謝及生長(zhǎng)定殖能力。將上述結(jié)果與相關(guān)文獻(xiàn)報(bào)道整合分析后得出,ermB陽(yáng)性彎曲菌在體內(nèi)競(jìng)爭(zhēng)初期發(fā)生的適應(yīng)性代價(jià)主要是由于運(yùn)動(dòng)能力下降及能量代謝失衡導(dǎo)致,而ErmB及鞭毛蛋白的高表達(dá)是影響能量平衡的關(guān)鍵因素。另外,研究還發(fā)現(xiàn)ermB陽(yáng)性彎曲菌可通過自身的調(diào)節(jié)恢復(fù)能量的攝取和利用水平,同時(shí)與細(xì)菌定殖、運(yùn)動(dòng)相關(guān)蛋白的二次代償可增強(qiáng)細(xì)菌繁殖能力,從而回復(fù)適應(yīng)性。綜上所述,ermB陽(yáng)性彎曲菌在我國(guó)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(yīng)性回復(fù)的現(xiàn)象有關(guān)。上述現(xiàn)象可能會(huì)使彎曲菌在獲得ermB基因后帶來(lái)更大范圍的流行傳播。本研究結(jié)果加深了我們對(duì)耐藥彎曲菌在流行傳播上的認(rèn)識(shí),同時(shí)為尋找控制耐藥彎曲菌傳播的方法提供了理論基礎(chǔ)。
[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(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】：2017
【分類號(hào)】：S852.61
本文編號(hào)：2343637
[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(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】：2017
【分類號(hào)】：S852.61
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 ;“基于濾膜通透性彎曲菌新檢測(cè)方法及彎曲菌超級(jí)耐藥島篩查技術(shù)”培訓(xùn)班會(huì)議紀(jì)要[J];疾病監(jiān)測(cè);2016年05期
2 張?chǎng)?佘丹陽(yáng);劉又寧;王睿;邸秀珍;梁蓓蓓;王悅;;細(xì)菌定量蛋白質(zhì)組學(xué)研究英文文獻(xiàn)計(jì)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(xué)研究中的分離分析技術(shù)及其研究進(jìn)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(duì)氟喹諾酮類和大環(huán)內(nèi)酯類藥物耐藥機(jī)制研究進(jìn)展[J];中國(guó)人獸共患病學(xué)報(bào);2013年09期
5 錢小紅;;定量蛋白質(zhì)組學(xué)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(biāo)記的蛋白質(zhì)組學(xué)定量方法研究進(jìn)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(xué)中的應(yīng)用[J];生物學(xué)雜志;2011年03期
8 楊國(guó)堂;李艷艷;張偉;孟令羽;;蛋白質(zhì)組學(xué)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(jìn)展[J];分析化學(xué);2010年03期
10 王華丙;張振義;包銳;陳宇星;;ABC轉(zhuǎn)運(yùn)蛋白的結(jié)構(gòu)與轉(zhuǎn)運(yùn)機(jī)制[J];生命的化學(xué);2007年03期
相關(guān)博士學(xué)位論文 前3條
1 鄧?guó)P如;耐藥基因島在我國(guó)部分地區(qū)動(dòng)物源和人源彎曲菌中的流行和傳播[D];中國(guó)農(nóng)業(yè)大學(xué);2016年
2 李會(huì);空腸彎曲菌酰胺醇類誘導(dǎo)耐藥株與敏感株的組學(xué)比較研究[D];中國(guó)農(nóng)業(yè)大學(xué);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(fēng)險(xiǎn)評(píng)估研究[D];中國(guó)農(nóng)業(yè)大學(xué);2014年
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