本文選題：大腸桿菌 + MLST分型　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：遏制細(xì)菌耐藥性行動計劃(2016-2020)是全民的共識與國家的意志的體現(xiàn),需要實施切實的技術(shù)措施。除了依據(jù)可靠的藥敏試驗結(jié)果精準(zhǔn)用藥外,噬菌體以其特異性強(qiáng)、與抗生素?zé)o交叉抗藥性、無殘留、對人畜無感染性、容易篩選獲得等獨特優(yōu)勢成為潛力巨大的選項。本文從山東省種雞場、商品雞場采集病料和糞樣篩選大腸桿菌的流行菌株,完善藥敏試驗方法,預(yù)警抗藥性菌株;篩選裂解性噬菌體,分析其生物學(xué)性質(zhì)和基因結(jié)構(gòu),通過注射、口服方式進(jìn)行治療試驗,以期控制種雞場致病性大腸桿菌的感染和流行。使用麥康凱培養(yǎng)基從臨床樣品和糞樣中篩選出疑似大腸桿菌,經(jīng)大腸桿菌特異性引物phoA共鑒定出大腸桿菌株117株。多位點序列分型(multilocus sequence typing,MLST)結(jié)果表明共有 65 個 ST 型,其中 ST155、ST156 與 ST69居多,另發(fā)現(xiàn)兩個新ST型。線性梯度平板能精確地測定MIC,推算藥物的原始濃度,能更好地保證質(zhì)控試驗的可靠性。當(dāng)按照美國臨床實驗室標(biāo)準(zhǔn)委員會(Clinical Laboratory Standards Institute,CLSI)或歐盟藥敏試驗委員會(European Committee on Antimicrobial Susceptibility Testing,EUCAST)的 S(Susceptible)、I(Intermediate)、R(Resistant)范圍標(biāo)準(zhǔn)設(shè)置0-S、S-R、R-8R這樣三個線性梯度,首先可以判斷菌株敏感性范圍及細(xì)菌群落精確的MIC值,并能分離出抗性菌株,預(yù)警該抗生素使用后可能出現(xiàn)的抗藥性,進(jìn)而指導(dǎo)臨床藥物的選擇和應(yīng)用。如果自動化生產(chǎn)即用型梯度平板,將便利臨床藥敏試驗,改進(jìn)抗藥性篩選技術(shù)。以半固體雙層平板噬菌斑法針對上述流行性耐藥大腸桿菌共分離純化到大腸桿菌噬菌體143株。挑選主要ST型菌株ST155對應(yīng)的3株大腸桿菌噬菌體進(jìn)行了測序和電鏡觀察,噬菌體均屬于有尾目、肌尾科T4噬菌體類型,由二十面體頭部與尾部組成,其中vB-EcolP-YSFl為新型噬菌體,與已測序噬菌體只有11%的相似性。vB-EcolP-XT4、vB-EcolP-RZⅢ與vB-EcolP-YSF1生物學(xué)特性分析表明,噬菌體效價均可達(dá)到10~9pfu/ml,且熱穩(wěn)定性較好,在60℃溫度下保溫1 h后噬菌體效價仍可達(dá)到10~7pfu/ml以上;最適生長溫度均在37 ℃左右;在pH值為4-10的區(qū)間內(nèi)能保持較好的裂解活性,最適pH偏中性;噬菌體的最佳感染復(fù)數(shù)一般在0.1-1之間,噬菌體在感染宿主菌后的潛伏期一般為20 min。通過大腸桿菌含有的毒力基因數(shù)目與其半數(shù)致死量確定攻毒菌株,以棋盤法試驗優(yōu)化最佳感染復(fù)數(shù),確定大腸桿菌噬菌體vB-EcolP-YSF1與大腸桿菌E.YSF-1肝作用的最佳濃度比,噬菌體治療采用胸肌注射肉雞,表現(xiàn)出了較好的治療效果,并隨著噬菌體效價的提高,肉雞的死亡率降低;噬菌體口服治療SPF雞實驗采用同時口服噬菌體vB-EcolP-RZⅢ與高抗的致病性大腸桿菌,噬菌體治療組比只口服致病菌組糞樣中該高抗的致病性大腸桿菌降低了至少1個數(shù)量級,這在實際生產(chǎn)條件下具有良好的臨床應(yīng)用價值,為后期噬菌體治療禽源大腸桿菌病奠定基礎(chǔ)。未來將根據(jù)上述試驗結(jié)果在種雞場進(jìn)行大腸桿菌大規(guī)模的環(huán)境凈化與大腸桿菌病防治試驗。
[Abstract]:The action plan to curb bacterial resistance (2016-2020) is the consensus of the whole people and the will of the country. It is necessary to implement practical technical measures. In addition to the accurate drug use according to the results of reliable drug sensitivity test, the bacteriophage is strong, no cross resistance to antibiotics, no residue, no infection to human and animal, and easy selection and so on. Advantage has become an option with great potential. In this paper, the epidemic strains of Escherichia coli were collected from the chicken farm of Shandong Province, the poultry farms were collected from the poultry farms and the feces samples were collected, the drug sensitivity test method was perfected, the resistant strains were early warning, the lysate phage was screened, the biological properties and the gene structure were analyzed, the treatment test was carried out by injection and oral administration, in order to control it. The infection and epidemic of pathogenic Escherichia coli in the chicken farm. The Escherichia coli was screened from clinical samples and feces by Makanke medium, and 117 Escherichia coli strains were identified by phoA specific primers. The results of multilocus sequence typing (MLST) showed that there were 65 ST types, including ST155, ST1. 56 and ST69 are many, and two new ST types are found. The linear gradient plate can accurately measure the MIC and calculate the original concentration of the drug. It can better guarantee the reliability of the quality control test. Timicrobial Susceptibility Testing, EUCAST) S (Susceptible), I (Intermediate), R (Resistant) range standard set 0-S, S-R, and such three linear gradients, first can determine the strain sensitivity range and the bacterial community accurate values, and can separate the resistant strain, early warning of the antibiotic may appear after the use of resistance, into the drug. To guide the selection and application of clinical drugs. If automated production is used as a gradient plate, the clinical drug sensitivity test will be facilitated and the resistance screening technology is improved. 143 strains of Escherichia coli are isolated and purified to the above epidemic resistant Escherichia coli by semi solid bilayer plate plaque method, and 3 of the main ST strains are selected for the selection of 3 of the main strains of Escherichia coli. The bacteriophages of Escherichia coli were sequenced and observed by electron microscope. The phage belonged to the phage of the caudate. The type of T4 phage was made up of the head and tail of the twenty facet body. The vB-EcolP-YSFl was a new phage, and only 11% of the phage had been sequenced.VB-EcolP-XT4, and the biological characteristics of vB-EcolP-RZ III and vB-EcolP-YSF1 were analyzed. The bacteriophage titer can reach 10~9pfu/ml, and the thermal stability is better. The phage titer can still reach more than 10~7pfu/ml after 1 h heat preservation at 60 C. The optimum growth temperature is about 37. The optimum cracking activity in the interval of pH value is 4-10, and the optimum pH is neutral. The best number of phage infection is generally between 0.1-1, The latent period of phage infection after infection is usually 20 min. through the number of virulence genes and half lethal dose of Escherichia coli to determine the virulence strain. The optimal number of infection is optimized by chessboard test, and the optimal concentration ratio of Escherichia coli vB-EcolP-YSF1 and Escherichia coli E.YSF-1 liver is determined, and phage therapy is used. The breast muscle injection of broilers showed a better therapeutic effect, and with the increase of phage titer, the mortality of broilers decreased, and phage SPF chickens were treated with oral phage vB-EcolP-RZ III and highly resistant pathogenic Escherichia coli, and the phage therapy group was more resistant to the pathogenic Escherichia coli in the fecal samples of the oral pathogenic bacteria group. The bacilli decreased at least 1 orders of magnitude, which was of good clinical value under actual production conditions and laid the foundation for the later phage treatment of avian Escherichia coli. In the future, large scale environmental purification and colibacillosis prevention and control test will be carried out in the chicken farm based on the experimental results.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.6

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