【摘要】：目的：本課題通過對銅綠假單胞菌臨床株和標(biāo)準(zhǔn)株的生物學(xué)特性、其生物被膜形態(tài)學(xué)的研究,探討了不同濃度的M331以及其聯(lián)合大環(huán)內(nèi)酯類抗生素對銅綠假單胞菌及其形成生物被膜的影響,以探討新的治療銅綠假單胞菌感染及它的生物被膜的方法；并初步探討了藥物M331的藥物毒性。 方法：應(yīng)用四區(qū)劃線法分離純化了菌株；通過對比其對細(xì)胞的粘附能力,來觀察其毒力；采用改良的組織培養(yǎng)平板法,建立了臨床株P(guān)A1和標(biāo)準(zhǔn)株P(guān)A27853生物被膜的模型；應(yīng)用微量稀釋法,測定了不同藥物及其聯(lián)合用藥對PA1、PA3、PA5、PA2059, PA27853的MIC值,應(yīng)用微量稀釋法,測定了PA1、PA2059的MBEC值；應(yīng)用MTT法測定了不同劑量M331及聯(lián)合用藥后PA1、PA27853生物被膜中的存活細(xì)菌數(shù)；應(yīng)用銀染法及掃描電鏡鑒定生物被膜,并觀察了不同劑量M331及其聯(lián)合用藥后PA1、PA27853的超微結(jié)構(gòu),對其菌落形態(tài)學(xué)進(jìn)行描述。 結(jié)果：臨床株和標(biāo)準(zhǔn)株不同菌株的產(chǎn)綠色素的情況不一；PA1、PA27853對Hela細(xì)胞的粘附性實(shí)驗(yàn)顯示,PA1、PA27853對Hela細(xì)胞的粘附類型為彌散性粘附,且臨床株P(guān)A1比標(biāo)準(zhǔn)株P(guān)A27853更易于粘附Hela細(xì)胞。銀染法快速鑒定結(jié)果顯示,都可見濃密黑染交織分布的物質(zhì),其間隙中可見聚集的短桿狀菌體。掃描電鏡觀察,銅綠假單胞菌呈短桿狀,聚集成團(tuán),為濃厚的粘液樣物質(zhì)緊密包繞,菌體之間相互以粘稠的纖維狀粘液絲相連。MTT法測定不同劑量M331及聯(lián)合羅紅霉素PA1、PA27853生物被膜中存活的細(xì)菌數(shù),結(jié)果顯示,羅紅霉素對M331有增效作用。應(yīng)用銀染法、掃描電鏡觀察不同劑量M331和聯(lián)合羅紅霉素處理的PA1、PA27853的生物被膜,可見隨著M331及羅紅霉素的劑量增大,PA1、PA27853生物被膜中的細(xì)菌逐漸被清除,細(xì)菌量逐漸減少。 結(jié)論：通過前期的實(shí)驗(yàn)研究,發(fā)現(xiàn)從貴州省耕土中分離的某種放線菌能產(chǎn)生一種抗生素,經(jīng)前期的實(shí)驗(yàn)證實(shí)其對銅綠假單胞菌療效較確定,對其它常見菌種無效,我們將此代謝產(chǎn)物命名為M331。本課題通過研究發(fā)現(xiàn),不同抗菌藥物對PA1、PA27853的浮游菌的殺菌活性存在明顯差異,M331的療效比慶大霉素弱,與多粘菌素B相當(dāng),強(qiáng)于羧芐青霉素。使用大環(huán)內(nèi)酯類抗生素能顯著增強(qiáng)M331的抗菌活性,酶抑制劑也能增強(qiáng)β-內(nèi)酰胺酶類抗生素的抗菌活性。國內(nèi)外研究發(fā)現(xiàn),14、15元環(huán)的大環(huán)內(nèi)酯類抗生素能抑制細(xì)菌藻酸鹽的合成,其作用機(jī)制可能是抑制藻酸鹽合成的關(guān)鍵酶——甘露糖脫氫酶(GMD)的活性,與其它藥物聯(lián)合應(yīng)用能夠抑制細(xì)菌生物被膜的形成。應(yīng)用不同劑量的M331及聯(lián)合羅紅霉素在生物被膜培養(yǎng)過程中分別處理PA1、PA27853,顯示了羅紅霉素聯(lián)合M331可有效抑制銅綠假單胞菌生物被膜的形成,其對Vero細(xì)胞無細(xì)胞毒性作用,是一個(gè)較為理想的針對銅綠假單胞菌的抗生素。
[Abstract]:Objective: to study the biological characteristics of Pseudomonas aeruginosa clinical and standard strains and the morphology of its biofilm. The effects of different concentrations of M331 and its combination with macrolide antibiotics on Pseudomonas aeruginosa and its biofilm formation were studied in order to explore a new method for the treatment of Pseudomonas aeruginosa infection and its biofilm. The drug toxicity of M331 was preliminarily discussed. Methods: the strain was isolated and purified by four-region crossing method, the virulence of the strain was observed by comparing its adhesion to cells, and the models of clinical strain PA1 and standard strain PA27853 biofilm were established by modified tissue culture plate method. The MIC values of different drugs and their combinations for PA1,PA3,PA5,PA2059, PA27853 were determined by microdilution method. The MBEC values of PA1,PA2059 were determined by microdilution method. MTT method was used to determine the number of viable bacteria in PA1,PA27853 biofilm after different doses of M331 and combined treatment. The biofilm was identified by silver staining and scanning electron microscope (SEM). The ultrastructure of PA1,PA27853 with different doses of M331 and its combination was observed and its colony morphology was described. Results: the green hormone production of different clinical strains and standard strains was different. The adhesion test of PA1,PA27853 to Hela cells showed that the adhesion type of PA1,PA27853 to Hela cells was diffuse adhesion, and PA1 was easier to adhere to Hela cells than the standard PA27853 strain. The results of rapid identification by silver staining showed that the interlaced substances with dense black dye could be seen, and the short rod-like bacteria could be found in the gap. Scanning electron microscope observation showed that Pseudomonas aeruginosa was a short rod shaped, agglomerated into a dense mucus-like material tightly wrapped around each other by a thick fibrous mucous filament. MTT method was used to determine the different doses of M331 and roxithromycin PA1,. The number of bacteria surviving in the PA27853 biofilm showed that roxithromycin had a synergistic effect on M331. Silver staining was used to observe the biofilm of PA1,PA27853 treated with different doses of M331 and roxithromycin. With the increase of the dose of M331 and roxithromycin, the bacteria in the biofilm of PA1,PA27853 were gradually eliminated and the amount of bacteria decreased. Conclusion: it was found that some actinomycetes isolated from cultivated soil of Guizhou province could produce an antibiotic. The experimental results showed that the effect on Pseudomonas aeruginosa was more definite, but not on other common strains of Pseudomonas aeruginosa. We named this metabolite M331. In this study, it was found that the bactericidal activity of different antimicrobial agents against PA1,PA27853 was significantly different. The efficacy of M331 was weaker than that of gentamicin and was similar to polymyxin B and stronger than that of carbenicillin. The antimicrobial activity of M331 was significantly enhanced by macrolides, and the antibacterial activity of 尾 -lactamases was also enhanced by enzyme inhibitors. Studies at home and abroad have shown that macrolides of 14 and 15 ring can inhibit the synthesis of bacterial alginate, and its mechanism may be the inhibition of the activity of mannose dehydrogenase (GMD), a key enzyme in the synthesis of alginate. Combination with other drugs can inhibit the formation of bacterial biofilm. The treatment of PA1,PA27853, with different doses of M331 and roxithromycin showed that roxithromycin combined with roxithromycin could effectively inhibit the formation of biofilm in Pseudomonas aeruginosa and had no cytotoxic effect on Vero cells. It is an ideal antibiotic against Pseudomonas aeruginosa.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378.991

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