【作者】 羊云飞；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2011， 博士

【摘要】 牦牛是藏区牧民的最重要生产和生活资料,牦牛的大肠杆菌病在牧区普遍存在。在其他地区随着抗生素的不断使用,大肠杆菌耐药性问题已越来越严重。但是在色达藏区,海拔在3500米以上,由于气候寒冷,路途遥远,条件简陋,采样极为不便,对牦牛、牧民的大肠杆菌耐药性及相关性的研究还未见报道。本论文针对藏区牦牛、牧民源大肠杆菌进行分离鉴定、耐药性的传播,对磺胺药、氟苯尼考耐药基因等问题,开展了系列研究工作,取得以下结果：1、藏区牦牛、牧民源大肠杆菌分离、血清学鉴定及16S rRNA分析从四川甘孜色达藏区9个不同乡镇的牧场,采集了具有牦牛210份粪便样品及同牧场的90份鼻腔拭子,进行采集、接种、保存条件摸索,对分离大肠杆菌进行生化学、血清学和致病性鉴定,抽取40株分离菌株作16sRNA分析。结果表明,采用80%甘油加30%脱脂牦牛奶作保护剂,可将接种成功率从10%提高到70%,菌种能保存到一周以上。共分离出株114牦牛源大肠杆菌和70株牧民源大肠杆菌。有98株牦牛源大肠杆菌菌的血清型被鉴定出来,分属24种血清型,其中有9种优势血清型占了60%的比例。有58株牧民源大肠杆菌的血清型被鉴定出来,分属12种血清型,其中有6种优势血清型占了62.8%的比例。鉴定出了22株牦牛源为致病性大肠杆菌,有20株的血清型鉴定出来,其中O78、O26、O111所占的比例最高,分别为18.2%、13.6%、13.6%,为致病性大肠杆菌的优势血清型。16S rRNA分析,这些细菌都与登录的大肠杆菌同源性为98-99%。进化树结果表明,四川甘孜色达藏区牧民源的与牧民源大肠杆菌聚为一类,牦牛源与牦牛源大肠杆菌也聚为一类。该研究首次建立了一套在高海拔、高寒条件下采样、分离、保存细菌的简单实用方法,该方法的建立为在当地开展细菌的科研工作铺下了的技术基石。鉴定了四川甘孜色达藏区牦牛致病性大肠杆菌的优势血清型。首次用16S rRNA鉴定了四川甘孜色达藏区牦牛、牧民的大肠杆菌,研究结果为进一步研究藏区牦牛源大肠杆菌提供了基础材料。2、四川甘孜色达藏区牦牛、牧民源大肠杆菌对15种常用抗生素的MIC测定及比较采用药物平板稀释法对四川甘孜色达藏区114株牦牛源和70株牧民源大肠杆菌进行15种抗菌药的MIC值测定,然后对结果进行的比较分析。结果表明,四川甘孜色达藏区牦牛源大肠杆菌对磺胺甲基异嗯唑、磺胺嘧啶、氟苯尼考的耐药性最高,分别为44%、40.4%、11.4%；对其他抗生素的耐药性低：分别为阿莫西林8.78%、氨苄西林6.14%、庆大霉素3.5%、链霉素1.75%；对新霉素、多西环素、土霉素、诺氟沙星、环丙沙星、萘啶酸、壮观霉素、头孢噻呋都敏感。四川甘孜色达藏区牧民源大肠杆菌对磺胺嘧啶和SMZ的耐药性最高,分别为57.1%和51.4%。而对其他抗生素的耐药性相对较低,从高到低分别氨苄西林22.9%、阿莫西林25.7%、链霉素20%、壮观霉素14.3%、庆大霉素11.4%、土霉素11.4%、头孢噻夫11.4%、萘啶酸11.4%、诺氟沙星8.57%、多西环素5.71%、氟苯尼考4.3%、环丙沙星2.86%、新霉素0%。比较牦牛源与牧民源大肠杆菌的耐药性发现：a、牧民源大肠杆菌的耐药性比牦牛源的比例高,种类多。b、两种来源的大肠杆菌都对磺胺药的耐药性在所有抗菌药中最高。c、在牧民不使用氟苯尼考的情况下,却检测出3株牧民源大肠杆菌对该药产生抗性。本研究首次对四川甘孜色达藏区的牧民、牦牛进行耐药性平行检测和比较分析数据,未见在同一环境中采样,对比分析牦牛与牧民源大肠杆菌耐药性的研究报道。该研究为治疗当地的大肠杆菌病提供了科学指导,为耐药性研究提供了更全面的资料。3、四川甘孜色达藏区牦牛、牧民源大肠杆菌对磺胺类和氟苯尼考耐药基因的PCR检测为检测四川甘孜色达藏区大肠杆菌主要耐药表型的耐药基因,在单重PCR方法的基础上,建立了检测磺胺耐药基因Sul1.Sul2和Sul3的三重PCR方法。利用此方法分别对50株牦牛源大肠杆菌的耐磺胺药物的基因进行检测,结果发现,在耐磺胺的50株菌中,基因阳性率为94%。其中携带基因型Sul1,Sul2,Sul1+Sul2的比例分别为36%,52%,6%。对40株牧民源大肠杆菌中,基因阳性率为92.5%。其中携带基因型Sul1,Sul2,Sul3,Sul1+Sul2,Sul1+Sul3的比例分别为42.5%,27.5%,7.5%,12.5%,2.5%。比较牦牛源与牧民源的磺胺耐药基因发现,牧民源大肠杆菌的磺胺药耐药基因型比牦牛的多两种,基因种类多Su13。本研究在国内首次对牧区人和动物磺胺耐药基因分布比较进行比较,为研究人与动物的耐药基因库提供资料。4、用脉冲场凝胶电泳PFGE对牦牛、牧民相同耐药基因型的菌株的分析研究为了研究同一个牧场里,牦牛和牧民之间是否存在耐药菌株的相互传播。本实验挑选26株耐药大肠杆菌,分为两组,每组13株菌,携带相同的耐药基因(Sull或flor),分离于两个牧场,来源不同宿主。采用脉冲场凝胶电泳(PFGE)进行分子分型和QantityOne软件进行菌株聚类分析。结果发现,在第一组携带磺胺耐药基因sull的13株大肠杆菌可分成12个PFGE谱型,其中含有一个克隆系,菌株牧民源C2与C4之间的相似系数达到0.9。在第二组,携带有氟苯尼考耐药基因(flor)13株菌可分成8个PFGE谱型,含有三个克隆系。其中一个克隆系里牧民源菌株B3与牦牛源菌株B12、B13具有0.88和0.93的相似系数。这些数据表明了在同一个牧场内,牧民与牧民、牦牛与牧民、牦牛与牦牛之间都存在着相同PFGE分子型的相同耐药谱菌株,提供了耐药菌株水平传播的线索。与国内外的其它类似的研究比较,本实验在所选的对象上具有独特性,首次采用PFGE研究在藏区特殊生产和生活模式下的人和牛的耐药性关系,为保障牦牛产品安全和人类健康提供科学依据。5、携带磺胺耐药基因和氟苯尼考耐药基因大肠杆菌的转化试验和接合性试验挑选有特别耐药性的19株菌进行分组。A组：牦牛源大肠杆菌株7株,携带有flor基因,对磺胺甲基异嗯唑敏感。B组：牧民源大肠杆菌12株,携带有Sull或Sul2基因,对氟苯尼考敏感。对所有细菌进行质粒提取,电泳分析质粒图谱；采用Ca2+转化法把各菌株的质粒转化到受体菌E.coli JM109中,然后挑选转化子,测MIC值,提取质粒,用PCR检测耐药基因；将所有菌与受体菌E.coli C00进行接合试验,并测定了产生接合子的时间。结果表明,每株菌都检测到多条大小不等的质粒DNA条带,质粒获得率为100%,质粒分子量为0.4×103～90.0×103bp。在A组7类质粒中,有4类质粒转化成功；在B组12类质粒中有6类质粒成功转化；总的转化率为52.6%。在接合试验中,发现质粒转化不成功的菌株都没有与受体菌接合成功,在含耐氟苯尼考耐药基因质粒的4株牦牛源大肠杆菌中有2株接合成功；在含耐磺胺药基因质粒的6株牧民源大肠杆菌中有5株接合成功：发生接合转移的时间为3h～6h；总的接合率为36.8%。本实验在实验室证明了耐药性能通过质粒的转化和细菌的接合传播,揭示了耐药性通过质粒传播的比例与速度,为控制耐药性的传播提供了科学资料。同时,鉴定到了2株含有携带flor基因质粒的牦牛源供体菌A12、A17,携带Sul1、Sul2基因质粒的5株牦牛源供体菌B1、B7、H1、D3, H5,为进一步开展天然大肠杆菌耐药质粒传递的研究提供了素材。本研究在四川甘孜色达四川甘孜色达藏区对分离自牦牛、牧民的大肠杆菌进行了致病性鉴定、血清学鉴定、MIC值检测,检测了主要耐药品种的耐药基因,用PFGE分析了同牧场中牦牛与牧民之间耐药菌株的关系,检测了耐药质粒对耐药性的传递特性,为指导当地藏民科学用药,有效防治大肠杆菌等细菌性疾病,保障牦牛产品安全,防制耐药性泛滥,保护人类健康提供了科学依据。更多还原

【Abstract】 Because of adverse weather condition, distant far-flung and poor condition in Serta County with high altitude and extremely cold climate, there is no study on E. coli of Yak and herdsman in Serta County.Yaks are the most important to production material and living material for Tibetans. Yak E. coli disease is widespread in the pastoral area. As antibacterial is used widely, the problem of drug-resistance increased seriously. Because of the transmission of resistance, it was difficult to cure E. coli disease clinically. In order to get current state of resistance for yak and herdsman in Serta County Tibetans, and the mechanism of resistance transmission, the works had been launched as follows:1. E.coli isolated and serotype identification and 16S rRNA analysis210 samples of yaks faces and 90 human nasal swab were collected from 9 pastures in Serta County Plateau. The conditions were selected for inoculum and conservation of stains. The isolates were identified by biologic character,serotype and pathogenicity, which of 40 stains were identified by 16sRNA. The results showed that survival rate could be raised from 10% to 80% by taking 80% Glycerin and 10% skimmed milk as protective agent.114 E.coli stains of yak and 70 E.coli stains of herdsman were isolated.The O serotype was identified by 165 kind’s single factor E.coli O antisera. Results showed that 98 stains were identified of yak in 114. The E.coli strains of yak fell into 24 serotype groups of which the O101 (12.3%)、O2 (9.6%)、O68 (7.0%). O132 (7.0%)、O78 (5.3%)、O171 (5.3%)、O22 (4.4%)、O142 (4.4%)、O165 (4.4%) were predominant, accounted for 60%.58 stains were identified of herdsman in 70. E.coli strains of herdsman fell into 12 serotype groups of which the O81 (14.3%)、 030 (11.4%).05(11.4%)、0165(8.6%)、0132(8.6%)、O101(8.6%)were predominant, accounted for 62.8%. Serotype of yak was more than herdsman. And there was 7 same serotype between yak and herdsman, including 0132、O101、O15、08、078、0171、017, accounted for 37.5%(13/70). The pathogenicity of stains was checked by challenge assays for mice. Results showed that 22 strains were pathogenic to mice identified.20 strains isolated were identified by O serotype, but 2 strains were not. O78、O26、O111 were accounted for 18.2%、13.6%、13.6%, respectively. The serotype of 078、O26、0111 were predominant of pathogenic E.coli, accounted for 50%. The serotype of pathogenic E.coli of yak was identified firstly, which was important to produce vaccine for pathogenic yak E. coli.40 stains from herdsman and yak were amplified for 16S rRNA and sequenced. All sequences of 40 stains were blasted in GenBank. Results showed that homology of the isolates were 98-99% with other E.coli. And phylogenetic tree was constructed. Results showed that stains between herdsmen clustered in the same group and that stains between yaks clustered in the same group, too. The results showed the genetic relationship of E.coli from Tibetan Area and others.2. MIC testing and comparation of E.coli of yak and herdsman to 15 antibiotic in common use.The MIC of 114 strains of yak and 70 strains of herdsman from 9 pastoral areas were examined for their susceptibility to 15 antimicrobial agents by drugs dilution-plate method. The results of antimicrobial susceptibility test were analyzed according to the standard procedure advised by NCCLS. The results showed that sufisomezole, sulfadiazine and florfeniol may be the most resistant antimicrobial agents tested in this study of yak, accounted for 44%、40.4%、11.4%, respectively. The resistant rate of stains from yak was low to other antibiotics. The resistant rate from high to low was amoxicillin (8.78%)、ampicillin(6.14%)、gentamicin(3.5%)、streptomycin(1.75%).All stains from yak were 100% sensitive to neomycin, doxycycline, terramycin, norfloxacin, ciprofloxacin, nalidixic acid, spectinomycin, ceftiofur sodium. The resistant rate of stains from herdsman was 57.1% and 51.4% to sulfadiazine and SMZ, respectively. The followed antimicrobial agents were ampicillin(22.9%), amoxicillin(25.7%), streptomycin(20%), spectinomycin(14.3%), terramycin(11.4%), ceftiofur sodium(11.4%), norfloxacin(8.57%), doxycycline(5.71%), florfeniol(4.3%), ciprofloxacin(2.86%), Neomycin(0%). In summary, by comparing resistance of yak with herdsman, we can draw the conclusions:1. the resistance of herdsman was more serious than yak’s in this study; 2. the sulfonamide-resistance was highest in all the antimicrobial agents; 3. the florfeniol-resistance was detected in 3 strains of herdsman, while florfeniol was prohibited in human to treatment of the diseases.The results of drug sensitivity test of E.coli from Serta County were help to cure E. coli disease. At the same time, the results were useful to parallel examination and data analysis for yak and herdsman, understand the problem of resistance overall.3. Detection of PCR for sulfonamide-resistance genes and florfeniol-resistance gene of E.coli of yak and herdsmanIn order to detected resistance gene of E.coli in Sertar County in Tibetan pastoral area, the multi-PCR for Sul1, Sul2 and Sul3 gene and simple PCR for flor gene were established. By the multi-PCR method,50 strains of yak and 40 strains of herdsman sulfonamide-resistant isolates were checked. Results showed that sulfonamide-resistance was different from yak and herdsman. The Sul2 (>50%) gene of sulfonamide-resistant isolates from yak was predominant, and sul2> sul1> sul3. The sul3 gene was no identified in isolates of E. coli from yak. In 50 isolates, more than one gene coding for sulfonamide resistance was present:sul1 and sul2 in three.The Sul1 (>40%) gene of sulfonamide-resistant isolates from herdsman was predominant, and sul1> sul2> sul3. The sul3 gene was also identified in 3 isolates of E. coli from herdsman. In 40 isolates, more than one gene coding for sulfonamide resistance was present:sul1 and sul2 in five; sul1 and sul3 in one. Sulfonamide-resistant genes of herdsman were more complicated than yak’s.16 stains were detected by the simple PCR method of flor gene. Results showed that 14 (87.5%) flor genes was detected in 16 florfeniol-resistant isolates from yak,3(100%) flor gene was detected in 3 florfeniol-resistant isolates from herdsman.4. The study on genetic relation of E. coli of yak and herdsman with the same resistance gene by PFGETo investigate the dissemination of resistance between yak and herdsman in the same Serta County ranch.26 resistant E.coli isolated from two ranch and different host were divided into two groups, and each group had 13 stains with the same similar resistance gene(Sull or flor) profiles were characterized by pulsed-field gel electrophoresis (PFGE) after XbaI digestion by using the CHEF system of Bio-Lab. Software Qantity One was used to analyze genetic relationship study between stains. The results showed that 13 E.coli strains with sul1 gene were classified into 12 different subtypes in the first group. An E.coli clone was detected, and similarity index was up to 0.9 between C2 and C4 from herdsman. In the second group,13 E.coli strains with flor gene were classified into 8 different subtypes. Three E.coli clones were detected, and similarity index was up to 0.88 and 0.93 between B3 and B12、B13 from yak,respectively. The PFGE patterns of strains suggested that transmission of resistant E. coli clones between herdsman and herdsman, as well as yak and herdsman.The resistance relationship between yak and herdsman in Serta County ranch was analyzed for the first time. And the results of transmission of resistant E.coli clones between yak and herdsman were important evidence on managing antibiotics of animal strictly, and preventing resistance widespread.5. Transformation test and conjugation test of E.coli of yak and herdsman with the same sulfonamide-resistance genes or florfeniol-resistanceThe 19 resistant strains were divided into two groups. A group:the 7 strains from yak with flor gene were susceptive to Sulfamethoxazole. B group:the 12 strains from herdsman with Sull or Sul2 gene were susceptive to Florfeniol. Plasmid DNA was purified by Plasmid MiniPrep Kit of TianGen Company. The plasmid profile was analyzed by agarose gel electrophoresis. Gel electrophoresis analysis demonstrated the presence of many visible plasmid bands from every isolate. Plasmids were discovered in all E.coli strains. Each profile contained more than one kind of plasmid. E.coli strains contained maximum plasmid was 10, and minimum plasmid was 2. The molecular weight of plasmids was from 0.4×103 to 90.0×103bp.19 kind plasmids were transformed into E.coli JM109 as recipient by Ca2 transformation. Then transformants was selected, plasmid DNA was extracted, MIC was measured and resistant genes were amplified by PCR. Results showed that the 7 kinds of plasmids with florfeniol-resistance gene from yak were transformed into E.coli JM109 separately.3 kinds of them were transformed successfully. While, The 12 kinds of plasmids with sulfonamide-resistance gene from herdsman were transformed into E.coli JM109 separately.6 kinds of them were transformed successfully. Transformation rate was 52.6% generally。The strains which could be transformed as donor were conjugated into E.coli C00 as recipient. Generated time of transconjugants was measured. Results showed that 2 strains in 4 with florfeniol-resistance from yak were conjugated into E.coli C00 successfully. And plasmid with flor gene was extracted from donor strain A12 and A172.5 strains in 6 with sulfanilamide-resistance from herdsman were conjugated into E.coli C00 successfully. And plasmid with sul1 gene was extracted from donor strain B1、B7 and H1,while sul2 gene from donor strain D 3and H5. The time of conjugation was from 3 to 6 hour. Transconjugants rate was 70% generally。On the one hand, the study revealed transmission of resistance by plasmid transformation and plasmid conjugal transfer, the transmission ratio and speed, which was important to provide the information on controlling transmission of resistance. On the other hand, plasmids with flor gene were identified from donor strain A12 and A172, and plasmids with sul1 or sul2 gene were identified from donor strain B1, B7, H1, D3 and H5,which would help to study on transmission of resistance between wildtype E.coli. The study on transmission of resistance plasmid systematically had not been reported in china Base on the special Tibetan pastureland, pathogenicity, serotype and MIC of E.coli isolates were indentified, which were beneficial to supply evidence to cure E.coli disease and guide the local civil and industrial needs. In the study, the resistance gene was checked from phenotypic resistance strains; genetic relationship was analyzed between yak and herdsman; and the character of transmission of resistance by plasmid was checked. The results were useful to manage antibiotics for animals strictly,E. coli of yak and herdsman collected from Sertar County in Tibetan closure pastoral area in this study. Transmission of resistant E.coli clones between yak and herdsman was proved by PFGE. The character of transfer of resistance to medicinal was studied by plasmid conjugation. The results were useful to manage antibiotics for animals strictly and protect human health and sanitation.更多还原