携带ermS基因畜禽源弯曲菌的流行及适应性机制的研究

发布时间：2018-11-19 22:31

【摘要】：空肠弯曲菌(C.jejuni)和结肠弯曲菌(C.coli)是人源及动物源中传播范围较广、致病性较强的食源性病原菌。大环内酯类药物是治疗弯曲菌感染的首选抗菌药物之一。近年来,由于抗菌药物的大量使用,弯曲菌对大环内酯类药物的耐药率呈明显上升趋势,而介导大环内酯类耐药的核糖体甲基化酶基因ermB在弯曲菌中的出现,给有效控制和治疗耐药弯曲菌带来很大挑战。另外,ermB基因稳定的水平传播特性也可能会导致耐红霉素弯曲菌的大范围流行。因此,本研究旨在调查ermB基因近年来在中国地区的流行及传播特征,并在此基础上进一步研究携带ermB基因弯曲菌的适应性变化特点和相关机制。本研究于2015年在山东、上海、广东地区采集到的1134份猪源(n=544)、鸡源(n=590)粪便或盲肠内容物样本中共分离得到370株弯曲菌(分离率:32.6%),运用PCR对进行ermB基因检测共得到37株ermB阳性C.coli,阳性率为10.0%。结合2013与2014年的数据分析,2013～2015年间ermB基因的阳性分离率分别为2.1%、8.2%、10.0%。而山东、上海、广东三个地区在三年间的总阳性率分别为1.1%、3.3%、15.1%。广东地区流行率较高,且呈现明显上升特点。上海及广东地区的ermB阳性菌株表现出较高的同源性,并出现了与2011年分离自人源ermB阳性菌高度相似的谱型,推测优势克隆型己在动物源与人源之间进行跨地域、跨时间的克隆传播。鉴于ermB阳性弯曲菌在我国部分地区的流行率呈上升的特征,推测ermB基因可能导致弯曲菌在适应性方面产生变化。为了解其适应性变化特点,本研究构建了两对遗传背景一致的ermB耐药工程菌株,并进行体内、体外试验。体内、体外生长试验结果均表明,携带ermB的菌株生长、定殖能力上较敏感菌相比均出现下降;而在体内竞争试验中,ermB菌株在接种初期表现出适应性代价,而在接种后期发生适应性回复现象。代偿菌株经体内体外生长、竞争的验证试验后,确证其出现了一定的适应性回复。为探索这一不同的适应性变化机制,尝试运用定量蛋白质组学方法研究发生适应性改变时弯曲菌在蛋白水平上的差异变化。运用同重同位素标签标记定量法(iTRAQ)进行筛选,耐药菌相对于敏感菌有123个差异显著蛋白,而代偿菌相对于耐药菌有25个差异显著蛋白。同时还运用了双向荧光差异凝胶电泳法(2D-DIGE)筛选,在代偿菌中发现13个与耐药菌相比差异表达的蛋白。通过GO、Pathway、COG等分析,这些差异蛋白主要影响了细菌的能量代谢及生长定殖能力。将上述结果与相关文献报道整合分析后得出,ermB阳性弯曲菌在体内竞争初期发生的适应性代价主要是由于运动能力下降及能量代谢失衡导致,而ErmB及鞭毛蛋白的高表达是影响能量平衡的关键因素。另外,研究还发现ermB阳性弯曲菌可通过自身的调节恢复能量的摄取和利用水平,同时与细菌定殖、运动相关蛋白的二次代偿可增强细菌繁殖能力,从而回复适应性。综上所述,ermB阳性弯曲菌在我国部分地区的流行呈上升特征,且有可能已在人畜间发生相互传播,其原因除了抗菌药物的筛选压力外,还可能与携带ermB基因菌株存在适应性回复的现象有关。上述现象可能会使弯曲菌在获得ermB基因后带来更大范围的流行传播。本研究结果加深了我们对耐药弯曲菌在流行传播上的认识,同时为寻找控制耐药弯曲菌传播的方法提供了理论基础。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.61

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