本文選題：假絲酵母菌 + 氟康唑��； 參考：《南昌大學(xué)》2010年碩士論文

【摘要】： 第一部分假絲酵母菌的分離、鑒定與藥敏分析 目的: 對引起假絲酵母菌感染的臨床菌株進(jìn)行分離鑒定,研究江西省假絲酵母菌臨床分離株的流行病學(xué)特點,并監(jiān)測其對氟康唑藥物的耐藥性,為合理使用抗生素提供依據(jù)。 方法: 對2005-2008年收集的2681株假絲酵母菌臨床分離株,采用CHROMagar顯色培養(yǎng)基、Vitek2 YST鑒定條和假絲酵母菌基因快速診斷-多重PCR鑒定法進(jìn)行菌株鑒定,并按照美國國家臨床試驗標(biāo)準(zhǔn)化研究所(CLSI)推薦的《酵母菌的液基稀釋法抗真菌藥物敏感試驗參考方案第二版》(M27-A2)方案測定2681株假絲酵母菌對氟康唑藥物的敏感性。 結(jié)果: 2005-2008年共收集假絲酵母菌2681株,其中,白假絲酵母菌1903株,占71.0%;光滑假絲酵母菌486株,占18.1%;熱帶假絲酵母菌142株,占5.3%;克柔假絲酵母菌99株,占3.7%;其他菌種為51株,占1.9%。其中,1690(63.0%)株來源于痰液,623 (23.2%)株來自陰道,169(6.3%)株分離自尿液,102(3.8%)株來自大便,44(1.6%)株分離于血液,10(0.4%)株分離于腹水,其余43(1.6%)株來源其他部位。白假絲酵母菌在痰、陰道、大便、腹水、尿液和血液中所占的比例分別為:76.1%、68.2%、63.7%、60%、47.3%和31.8%;光滑假絲酵母菌在痰、陰道、大便、腹水、尿液和血液中所占的比例分別為:14.7%、19.7%、22.5%、40.0%、34.9%和40.9%;熱帶假絲酵母菌在尿液中所占的比例為12.4%,而在痰、陰道、大便、血液中所占的比例不到7.0%。 2681株假絲酵母菌中,氟康唑耐藥株為226株,耐藥率為8.4%;氟康唑劑量依賴敏感株109株,占4.1%;敏感株2346株,敏感率為87.4%。白假絲酵母菌、光滑假絲酵母菌、熱帶假絲酵母菌和克柔假絲酵母菌的氟康唑耐藥率分別為4.5% (86/1903)、6.4% (31/468)、7.0% (10/142)和100% (99/99)。不同部位的假絲酵母菌的氟康唑耐藥率也有所不同,其中來自陰道部位的假絲酵母菌氟康唑耐藥率最高,為13.5% (84);其次為大便9.8% (10),尿液8.3% (14),其他部位7.0% (3),血液6.8% (3)和痰液6.6%(112)。 結(jié)論: 1、白假絲酵母菌仍然為臨床上最為常見和最主要的致病菌,其次為光滑假絲酵母菌。 2、白假絲酵母菌主要常見于呼吸道、陰道和腸道的感染,而光滑假絲酵母菌和熱帶假絲酵母菌主要見于無菌部位(血液和尿道)的感染�，F(xiàn)今假絲酵母菌的臨床感染譜已發(fā)生變化,其中非白假絲酵母菌引起的感染日趨增加,已成為血液和尿道真菌感染的主要原因。 3、假絲酵母菌江西臨床分離株對氟康唑總體耐藥率不高,血液與痰來源的假絲酵母菌對氟康唑的敏感性高,但是感染陰道部位的假絲酵母菌對氟康唑耐藥率高,達(dá)13.5%。 4、氟康唑依然是一個有效的抗真菌藥物,但對克柔假絲酵母菌和陰道假絲酵母菌的感染應(yīng)謹(jǐn)慎選擇合適的抗真菌藥物。 第二部分白假絲酵母菌耐藥基因ERG11突變分析 目的: 檢測白假絲酵母菌臨床分離株氟康唑靶酶編碼基因(ERG11)突變,探討其與耐藥性的關(guān)系。 方法: 以2005-2008年收集的白假絲酵母菌臨床分離株72株(氟康唑耐藥菌株49株,氟康唑劑量依賴敏感(S-DD)株16株和敏感菌株7株)為研究對象,PCR擴增白假絲酵母菌ERG11全基因序列,DNA測序后經(jīng)生物信息學(xué)比對及分析ERG11基因的突變。 結(jié)果: 經(jīng)DNA測序分析后,72株白假絲酵母菌ERG11基因序列中共發(fā)現(xiàn)27個無義突變和14個錯義突變,其中: (1) D225H、K342R、G450E和V488I這四個錯義突變僅見于氟康唑耐藥菌株;(2) G129A僅見氟康唑劑量依賴敏感株中;(3) Y132H、A114S、Y257H、V437I、G465S、G448E和K128T這七個突變同時出現(xiàn)于耐藥株及劑量依賴敏感株中。 此外發(fā)現(xiàn)在65株氟康唑耐藥株和劑量依賴敏感株中存在7種多位點錯義突變模式:(1) D116E、K128T、Y132H和G465S同時出現(xiàn)在2株耐藥株和3株S-DD株中;(2) A114S和Y257H同時出現(xiàn)在11株氟康唑耐藥株和3株S-DD株中,但不伴有其他位點的同義突變;(3) Y132H和G450E同時出現(xiàn)在2株氟康唑耐藥菌株中,但不伴有其他位點的同義突變;(4) Y132H和G448E同時出現(xiàn)在3株菌株中但不伴有其他位點的同義突變;(5) E266D和V488I同時出現(xiàn)在3株耐藥株中;(6)D116E、E266D和V488I同時出現(xiàn)在2株耐藥菌株中;(7)D116E、E266D和V437I同時出現(xiàn)在2株耐藥菌株和1株S-DD株中。 另外,我們也發(fā)現(xiàn)單突變模式比如V437I和K342R也存在于耐藥菌株中。 結(jié)論: 1.白假絲酵母菌敏感菌株和耐藥菌株ERG11基因的突變各不相同,多位點錯義突變模式和單位點錯義突變是導(dǎo)致耐藥的分子基礎(chǔ),多位點錯義突變是劑量依賴敏感株和耐藥株中ERG11基因突變的主要形式,也可能是導(dǎo)致耐藥的主要機制之一。 2.首次報導(dǎo)D116E、K128T、Y132H和G465S,D116E、E266D和V437I及D116E、E266D和V488I等多位點錯義突變模式。
[Abstract]:Part 1 isolation, identification and drug sensitivity analysis of Candida albicans
Objective:
The clinical isolates of Candida infection were isolated and identified, the epidemiological characteristics of Candida strains in Jiangxi province were studied, and the drug resistance to fluconazole was monitored to provide the basis for rational use of antibiotics.
Method:
The clinical isolates of 2681 Candida albicans collected in 2005-2008 years were identified by CHROMagar color culture medium, Vitek2 YST identification strip and the rapid diagnosis of Candida albicans gene multiple PCR identification, and the antifungal drug sensitivity of the liquid based dilution method of Saccharomyces cerevisiae, recommended by the National Institute for clinical trials of the United States (CLSI). The sensitivity of the 2681 version of Candida albicans to fluconazole was determined by the second version of the reference scheme (M27-A2).
Result:
2681 strains of Candida albicans were collected in the past 2005-2008 years, of which 1903 strains of Candida albicans, 71% Candida albicans, 18.1% of Candida smooth, 5.3% Candida tropics, 99 Candida Candida, 3.7%, 51, 1.9%., 1690 (63%) from the sputum, and 1903 strains from the vagina From urine, 102 (3.8%) strains were derived from stool, 44 (1.6%) isolated from blood, 10 (0.4%) isolated from ascites and the rest 43 (1.6%). The proportion of Candida albicans in sputum, vagina, stool, ascites, urine and blood were 76.1%, 68.2%, 63.7%, 60%, 47.3% and 31.8%, and Candida smooth in phlegm, vagina, and stool. The proportion of ascites, urine and blood are 14.7%, 19.7%, 22.5%, 40%, 34.9% and 40.9%, and the proportion of Candida tropicalis in urine is 12.4%, and the proportion in phlegm, vagina, stool, and blood is less than 7.0%.
Of the 2681 strains of Candida, 226 strains were resistant to fluconazole, the resistance rate was 8.4%, the dose of fluconazole was 109, 4.1%, and 2346 of the sensitive strains of Candida albicans 87.4%., Candida smooth Candida, Candida tropics and Candida krou were 4.5% (86/1903), 6.4% (31/468), 7, respectively. % (10/142) and 100% (99/99). The rates of fluconazole resistance in different parts of Candida were also different, among which the rate of fluconazole resistance from the vaginal Candida was the highest, 13.5% (84), followed by 9.8% (10), 8.3% (3) in urine, 7% (3), 6.8% (3) and 6.6% (112) in the sputum.
Conclusion:
1, Candida albicans remains the most common and most important pathogen in clinical practice, followed by Candida tropicalis.
2, Candida albicans are mainly common in respiratory, vaginal and intestinal infections, while Candida smooth and Candida tropicalis are mainly found in the sterile site (blood and urethra). The clinical infection spectrum of Candida albicans has changed, and the infection caused by Candida non Candida is increasing, and it has become a blood and a blood. The main cause of urethral fungal infection.
3, the resistance rate of Candida albicans Jiangxi clinical isolates to fluconazole was not high. The sensitivity of Candida albicans to fluconazole was high in blood and sputum sources, but the resistance rate of Candida infected vaginal Candida to fluconazole was high, up to 13.5%.
4, fluconazole is still an effective antifungal drug, but it should be carefully chosen for the infection of Candida kurou and Candida vagina.
The second part is ERG11 mutation analysis of drug resistance gene of Candida albicans.
Objective:
Objective to detect the mutation of the target gene (ERG11) of fluconazole in clinical isolates of Candida albicans, and to explore its relationship with drug resistance.
Method:
72 strains of Candida albicans collected in 2005-2008 years (49 strains of fluconazole resistance strain, 16 S-DD strain sensitive (S-DD) strain and 7 sensitive strains) were used as the research object. PCR amplification of Candida albicans ERG11 whole gene sequence, DNA sequencing after sequencing of bioinformatics and analysis of the mutation of ERG11 gene.
Result:
After DNA sequencing analysis, 27 non sense mutations and 14 missense mutations were found in 72 Candida albicans ERG11 gene sequence, of which four missense mutations (1) D225H, K342R, G450E and V488I were only found in fluconazole resistant strains; (2) G129A only observed the dose of fluconazole in the Lai Min strain; (3) Y132H, A114S, Y257H, V437I, G465S, G465S, etc. The seven mutations also occurred in drug-resistant strains and dose-dependent sensitive strains.
In addition, there were 7 multipoint missense mutations in 65 strains of fluconazole resistance and dose dependent sensitive strain: (1) D116E, K128T, Y132H and G465S appeared in 2 resistant and 3 S-DD strains; (2) A114S and Y257H appeared in 11 fluconazole resistant and 3 S-DD strains, but did not accompany the synonymous mutation of other loci; (3) Y132 H and G450E appeared at the same time in 2 strains of fluconazole resistant strains, but did not have synonymous mutations with other loci; (4) Y132H and G448E appeared in 3 strains but did not have synonymous mutations with other loci; (5) E266D and V488I appeared at the same time in 3 resistant strains; (6) D116E, E266D and V488I appeared in 2 resistant strains; (7) D116E, E266D V437I and S-DD were found in 2 resistant strains and 1 strains.
In addition, we found that single mutation patterns, such as V437I and K342R, were also found in drug-resistant strains.
Conclusion:
1. the mutation of the ERG11 gene of the sensitive and drug-resistant strains of Candida albicans is different. The multipoint missense mutation mode and the unit point missense mutation are the molecular basis for the resistance. The multipoint missense mutation is the main form of the ERG11 gene mutation in the dose dependent and drug-resistant strains, and may also be the main mechanism leading to the resistance. 1.
2. for the first time, D116E, K128T, Y132H and G465S, D116E, E266D and V437I, D116E, E266D and V488I were first reported.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R379

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