【作者】 刘英玉；

【导师】 何启盖；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2013， 博士

【摘要】 副猪嗜血杆菌(Haemophilus parasuis, HPS)是引起猪的多发性浆膜炎、脑膜炎和关节炎的一种革兰氏阴性杆菌。副猪嗜血杆菌病又称为Glasser’s病,给世界养猪业造成了巨大的经济损失。副猪嗜血杆菌具有15个血清型和不可分型菌株,有很强的宿主特异性,2周龄到4月龄的猪易感,尤其对早期断奶仔猪造成了严重的危害。目前主要通过灭活苗和抗生素来预防和控制副猪嗜血杆菌病,但单价疫苗免疫不能产生交叉保护作用,所以通过抗生素进行预防和治疗该病,仍是一种常见的方法。而抗生素的不合理使用导致越来越多的细菌对抗生素产生了耐药性,严重影响了抗生素的杀菌作用。近年来,许多国家都报道了副猪嗜血杆菌存在耐药现象,因此,有必要对副猪嗜血杆菌进行耐药性调查和耐药机制研究。本研究首先对临床菌株进行了耐药流行病学调查和内源性质粒的分离鉴定,然后分析青霉素结合蛋白的突变位点与氨苄青霉素耐药菌株和携带内源性质粒菌株之间的关系。同时,通过基因表达谱芯片研究副猪嗜血杆菌SH0165在亚抑制浓度和抑制浓度替米考星作用下的转录组学变化,揭示了亚抑制浓度和抑制浓度(0.25μg/mL和8μg/mL)替米考星的作用机制。现将主要研究结果报告如下：1.副猪嗜血杆菌的耐药流行病学调查和内源性质粒的分离鉴定本试验对2009年分离的92株副猪嗜血杆菌进行了10种常用抗生素的耐药流行病学调查,其中存在青霉素(21.74%)、氨苄青霉素(23.91%)、林可霉素(23.91%)、阿莫西林(15.22%)等耐药菌株,对头孢噻肟和氯霉素敏感的菌株,均为90.22%,而22种耐药基因检测存在氨基糖苷类耐药基因aadB有20株,四环素类耐药基因tetB有11株,磺胺类耐药基因sulll有8株。同时从92株副猪嗜血杆菌中分离出6株菌株携带有内源性质粒,并对其中5个内源性质粒进行了测序,质粒FZJ5839是隐性质粒,其余都是耐药质粒。质粒pHPS1019编码耐药基因tetB和复制蛋白rep;质粒FHN1020和质粒FZG1012是来自不同猪场的相同耐药质粒,编码了链霉素类耐药基因strA和磺胺类耐药基因sulll及MOB移动蛋白家族；质粒FJS5863编码了β-内酰胺类耐药基因blaRoB-1,氨基糖苷类双功能修饰酶基因aacA-aphD, MOB移动蛋白家族和复制蛋白rep,其中耐药基因aacA-aphD首次在副猪嗜血杆菌中报道。隐性质粒FZJ5839是测序中最大的质粒(11,812bp),编码了移动蛋白mobA_mobL,重组酶rec,复制蛋白rep和分裂蛋白parA。其中携带耐药质粒的菌株中只有耐药基因tetB不表现耐药表型,其余耐药基因均存在耐药表型。而对分离的内源性质粒进行耐药基因检测,其结果是不仅存在质粒编码的耐药基因,也存在耐药基因blaTEM和dhfrV等。通过对耐药质粒进行同源性分析发现与巴斯德菌属或嗜血杆菌属中耐药质粒进化关系较近。2.分析副猪嗜血杆菌青霉素结合蛋白的突变位点根据HPS SH0165的基因组信息,总结HPS青霉素结合蛋白(Penicillin-binding proteins, PBPs)的类型,主要包括6类PBPs,其中有4个HMM PBPs[2个A类PBPs (mrcA和pbp1B)和2个B类PBPs (ftsI和。ftsI-2)]和2个LMM PBPs (dacA和dacB)及1个多功能转糖酶mtgA和1个PBP3相关的羧基末端蛋白酶prc。通过琼脂稀释法对2009年选取的22株和2012年分离的34株HPS进行6种p-内酰胺类抗生素(青霉素,头孢噻肟,氨苄青霉素,苯唑西林,阿莫西林和克拉维酸)的耐药检测,存在氨苄青霉素和苯唑西林抗性的菌株达到95%以上,同时从2012年34株HPS中分离出2个内源性质粒。根据菌株是否对氨苄青霉素和苯唑西林耐药,是否携带内源性质粒来选取20株HPS进行PBPs的克隆测序(其中2009年和2012年各10株),参考菌株是HPS SH0165和HPS标准5型,测序结果PBPs基因是高度保守的。通过PBPs碱基序列的进化树关系,预测PBPs存在的突变位点,并在蛋白的三维结构上定位突变位点,分析发现PBPs肽基转移酶上的突变位点与p-内酰胺类抗生素耐药性有关,糖基转移酶上的突变位点与内源性质粒传递有关。在携带质粒FJS5863的菌株09FA17中发现mrcA基因在突变位点638处插入7个氨基酸ATENTTD,导致下游有11个突变位点,并在一定程度上改变了mrcA蛋白的三维结构,推测该突变与菌株携带质粒FJS5863编码的耐药基因blaROB-1有关。其中有8株菌的dacB基因在氨基酸C-端插入了199个氨基酸,推测与内源性质粒的传递有关。有5株菌的dacA基因在358氨基酸处突变为终止密码子。3.替米考星诱导副猪嗜血杆菌SH0165的转录组学研究为了揭示替米考星作用于]HPS SH0165的分子机制,根据HPS SH0165的基因组序列,设计了2052个探针合成基因表达谱芯片来研究不同浓度(亚抑制浓度0.25μg/mL和抑制浓度8μg/mL)的替米考星对HPS SH0165的转录组学变化,成功筛选到405个差异表达基因(p≤0.05,Fc≥1.5),这些基因涉及到热休克蛋白、核糖体蛋白、蛋白质的生物合成、细胞壁合成和细胞转运相关的过程。在0.25μg/mL和8μg/mL两种浓度组中既存在共同变化的基因也存在其特有的差异表达基因,其中共同上调表达的基因有56个和共同下调表达的基因有39个。在0.25μg/mL浓度组中检测出302个差异表达基因,在替米考星压力下正常的生长以调节ABC转运体和PTS系统中基因为特点,而在8μg/mL浓度组中检测出198个差异表达基因,因生长被完全抑制以下调表达RNA聚合酶和错配修复通路中基因为特点。芯片中差异表达基因的功能分析揭示了HPS SH0165主要通过调节蛋白的合成和胞膜转运来适应替米考星的作用,替米考星诱导HPS SH0165的转录谱可为研究细菌的生理功能提供新思路及为制定治疗策略提供新靶标。更多还原

【Abstract】 Haemophilus parasuis is the causative agent of Glasser’s disease in swine, charac-terized by fibrinous polyserositis, meningitis and polyarthritis, and it also caused great economic losses in the global pig industry.15H. parasuis serovars have been recognized based on immunodiffusion of heat-stable polysaccharide antigens, while some isolates are still non-type serovars. H. parasuis has strong host specificity. H. parasuis could easily infect the pigs ranging from2weeks to4months and can cause severe diseases to the pigs, especially for the early-weaned piglets. The prevention and control of H. parasuis disease can be achieved through vaccine immunization and antibiotics therapy. The vac-cines usually confer protection against challenge with homologous serovar, but the results of the cross-protection are variable. So antibiotics were used as the main prevention strategy. However abuse of antibiotics would encourage an increase in resistance to the antibiotics, decreasing the efficiency of them. In recent years, the drug resistance cases in H. parasuis have been reported in many countries. So research the resistance mechanism and investigating the drug resistance in H. parasuis is very important for us to control this disease. Firstly, this study investigated the epidemiology about the drug resistance, isola-tion and identification of endogenous plasmids in clinical isolates of H. parasuis. Sec-ondly, the mutation sites that penicillin binding proteins were analyzed to identify the mechanism of the resistance to ampicillin and the endogenous plasmids in clinical isolates. At the same time, a whole-genome DNA microarray was used to profile the transcription-al response of H. parasuis SH0165induced tilmicosin at sub-inhibitory and inhibitory concentrations.The principal results were presented as followed:1. Investigating epidemiology about the drug resistance in H. parasuis, isolation and identification of endogenous plasmidsOur study investigated the epidemiology about the drug resistance in H. parasuis to ten kinds of common antibiotics, and the endogenous plasmids were also isolated and identified based on the92strains isolated in2009. The results revealed that20strains (21.74%) were resistance to penicillin,22strains (23.91%) were resistance to ampicillin,22strains were resistance to lincomycin and14strains (15.22%) were resistance to amoxicillin, while83strains (90.22%) were sensitive to cefotaxime and chloramphenicol. The detected22resistance genes of the strains were aadB gene, tetB gene and sulll gene which included20strains,11strains and8strains, respectively. At the same time, six en- dogenous plasmids of H. parasuis were identified, and the genomes of five of them were sequenced. Beside the plasmid FZJ5839is a recessive plasmid, the other plasmids were the resistant plasmids. Plasmid pHPS1019encoded the tetracycline resistance gene tetB and the replication protein rep. Plasmid FHN1020and FZG1012encoded the streptomy-cin resistance gene strA and the sulfasulfonamide resistance gene sulll, the movement proteins MOB family, but they are the different sources of resistance plasmids. Plasmid FJS5863carried β-lactam resistance gene blaROB-1and aminoglycoside modification bi-functional enzyme genes aacA-aphD, encoded the mobile family proteins and the replica-tion protein rep. The recessive plasmid FZJ5839is one of the biggest plasmids, which had been sequenced (11,812bp), encoded the movement protein mobA_mobL, recombinase rec, replication protein rep and splitting protein par A. Antimicrobial agents sensitivity profiling revealed that strains were resistant to the streptomycin, sulfadimidine, gentami-cin, kanamycin and ampicillin, beside of the tetracycline. The detected resistance genes of the endogenous plasmids were presents the resistant genes blaTEM, dhfrV, strA and sulll. Finally, the resistance plasmids of H. parasuis were analyzed by the phylogenetic tree, we found that have high similarity with the resistance plasmids in Pasteurella and the re-sistance plasmids in Haemophilus.2. Analysis of genes encoding PBPs in clinical isolates of H. parasuisBased on the genomic sequence of H. parasuis SH0165, we summarized the PBPs types, including six classes of PBPs, which has four HMM PBPs [two A classes (mrcA and pbp1B) and two B classes (ftsl and ftsI-2)], two LMM PBPs (dacA and dacB), one monofunctional transglycosylase mtgA and one peptidoglycan carboxy-terminal protease prc. We investigated the H. parasuis isolates (including22strains isolated in2009and34strains isolated in2010) resistance to β-lactam antibiotics (penicillin, ampicillin, oxacillin, ceftiofur, amoxicillin, clavulanic acid) and found that the resistance strains to ampicillin and oxacillin have reached95%. At the same time, two endogenous plasmids of H. para-suis were isolated in2012. PBPs genes of twenty strains (each of ten strains in2009and in2012) that resistance to ampicillin or carried endogenous plasmid were sequenced. Then we compared them with the PBPs gene sequence of the H. parasuis SH065and the standard H. parasuis serotype5, which had been found that the gene was highly con-served among the strains. Analysis of genes encoding PBPs through the relationship of the evolutionary tree, we predicted that the PBPs genes mutation sites of these strain and located the mutation sites on the three dimensional structure of the PBPs. We predicted that the mutation sites of the transpeptidase in PBPs have related to the (3-lactam antibiot-ics of the resistance, the mutation sites of the transglycosylase in PBPs have related to the transfer of the endogenous plasmid in the bacteria. The mrcA gene had inserted seven amino acids (ATENTTD) in the mutation sites638of the FJS5863plasmid of09FA17strain. It has7amino acids insertion and11mutation sites in the downstream of the mrcA gene and causes the changes in the3D structure of the mrcA protein. We speculated that these variations were related to the plasmid FJS5863carried the resistance gene blaвов-1. The dacB gene of the eight strains had199amino acids insertion in the downstream of the stop codon, that this difference was related to the endogenous plasmids. Others, the dacA gene of the five strains had changed the amino acid located in the sites of358to encode the termination codon.3. Transcription profile of H. parasuis SH0165response to tilmicosinBased on the genomic sequence of H. parasuis SH0165,2052probes were designed and synthesied in situ microarray chip. To gain a more detailed understanding of the mo-lecular mechanisms underlying H. parasuis response to tilmicosin treatment, microarray technology was applied to analyze the variation in gene expression of isolated H. parasuis SH0165treated in vitro with sub-inhibitory (0.25μg/mL) and inhibitory (8μg/mL) con-centrations. Tilmicosin treatment induced differential expression of405genes (p≤0.05, Fc≥1.5), the encoded products of which are mainly involved in the heat shock response, protein synthesis, and intracellular transportation. The sub-inhibitory and inhibitory con-centrations of tilmicosin induced distinctive gene expression profiles of shared and unique changes, respectively. These changes included302genes mainly involved in protein ex-port and the PTS system to sustain cell growth, and198genes mainly related to RNA polymerase, recombination, and repair to inhibit cell growth. In silico analysis of func-tions related to the differentially expressed genes suggested that adaptation of H. parasuis SH0165to tilmicosin involves modulation of protein synthesis and membrane transport. Collectively, the genes comprising each transcriptional profile of H. parasuis response to tilmicosin provide novel insights into the physiological functions of this economically significant bacterium and may represent targets for future molecular therapeutic strate-gies.更多还原