临床假丝酵母菌药敏分析及耐药基因ERG11突变研究

发布时间：2018-04-26 07:49

本文选题：假丝酵母菌 + 氟康唑　；参考：《南昌大学》2010年硕士论文

【摘要】： 第一部分假丝酵母菌的分离、鉴定与药敏分析目的: 对引起假丝酵母菌感染的临床菌株进行分离鉴定,研究江西省假丝酵母菌临床分离株的流行病学特点,并监测其对氟康唑药物的耐药性,为合理使用抗生素提供依据。方法: 对2005-2008年收集的2681株假丝酵母菌临床分离株,采用CHROMagar显色培养基、Vitek2 YST鉴定条和假丝酵母菌基因快速诊断-多重PCR鉴定法进行菌株鉴定,并按照美国国家临床试验标准化研究所(CLSI)推荐的《酵母菌的液基稀释法抗真菌药物敏感试验参考方案第二版》(M27-A2)方案测定2681株假丝酵母菌对氟康唑药物的敏感性。结果: 2005-2008年共收集假丝酵母菌2681株,其中,白假丝酵母菌1903株,占71.0%;光滑假丝酵母菌486株,占18.1%;热带假丝酵母菌142株,占5.3%;克柔假丝酵母菌99株,占3.7%;其他菌种为51株,占1.9%。其中,1690(63.0%)株来源于痰液,623 (23.2%)株来自阴道,169(6.3%)株分离自尿液,102(3.8%)株来自大便,44(1.6%)株分离于血液,10(0.4%)株分离于腹水,其余43(1.6%)株来源其他部位。白假丝酵母菌在痰、阴道、大便、腹水、尿液和血液中所占的比例分别为:76.1%、68.2%、63.7%、60%、47.3%和31.8%;光滑假丝酵母菌在痰、阴道、大便、腹水、尿液和血液中所占的比例分别为:14.7%、19.7%、22.5%、40.0%、34.9%和40.9%;热带假丝酵母菌在尿液中所占的比例为12.4%,而在痰、阴道、大便、血液中所占的比例不到7.0%。 2681株假丝酵母菌中,氟康唑耐药株为226株,耐药率为8.4%;氟康唑剂量依赖敏感株109株,占4.1%;敏感株2346株,敏感率为87.4%。白假丝酵母菌、光滑假丝酵母菌、热带假丝酵母菌和克柔假丝酵母菌的氟康唑耐药率分别为4.5% (86/1903)、6.4% (31/468)、7.0% (10/142)和100% (99/99)。不同部位的假丝酵母菌的氟康唑耐药率也有所不同,其中来自阴道部位的假丝酵母菌氟康唑耐药率最高,为13.5% (84);其次为大便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)。结论: 1、白假丝酵母菌仍然为临床上最为常见和最主要的致病菌,其次为光滑假丝酵母菌。 2、白假丝酵母菌主要常见于呼吸道、阴道和肠道的感染,而光滑假丝酵母菌和热带假丝酵母菌主要见于无菌部位(血液和尿道)的感染。现今假丝酵母菌的临床感染谱已发生变化,其中非白假丝酵母菌引起的感染日趋增加,已成为血液和尿道真菌感染的主要原因。 3、假丝酵母菌江西临床分离株对氟康唑总体耐药率不高,血液与痰来源的假丝酵母菌对氟康唑的敏感性高,但是感染阴道部位的假丝酵母菌对氟康唑耐药率高,达13.5%。 4、氟康唑依然是一个有效的抗真菌药物,但对克柔假丝酵母菌和阴道假丝酵母菌的感染应谨慎选择合适的抗真菌药物。第二部分白假丝酵母菌耐药基因ERG11突变分析目的: 检测白假丝酵母菌临床分离株氟康唑靶酶编码基因(ERG11)突变,探讨其与耐药性的关系。方法: 以2005-2008年收集的白假丝酵母菌临床分离株72株(氟康唑耐药菌株49株,氟康唑剂量依赖敏感(S-DD)株16株和敏感菌株7株)为研究对象,PCR扩增白假丝酵母菌ERG11全基因序列,DNA测序后经生物信息学比对及分析ERG11基因的突变。结果: 经DNA测序分析后,72株白假丝酵母菌ERG11基因序列中共发现27个无义突变和14个错义突变,其中: (1) D225H、K342R、G450E和V488I这四个错义突变仅见于氟康唑耐药菌株;(2) G129A仅见氟康唑剂量依赖敏感株中;(3) Y132H、A114S、Y257H、V437I、G465S、G448E和K128T这七个突变同时出现于耐药株及剂量依赖敏感株中。此外发现在65株氟康唑耐药株和剂量依赖敏感株中存在7种多位点错义突变模式:(1) D116E、K128T、Y132H和G465S同时出现在2株耐药株和3株S-DD株中;(2) A114S和Y257H同时出现在11株氟康唑耐药株和3株S-DD株中,但不伴有其他位点的同义突变;(3) Y132H和G450E同时出现在2株氟康唑耐药菌株中,但不伴有其他位点的同义突变;(4) Y132H和G448E同时出现在3株菌株中但不伴有其他位点的同义突变;(5) E266D和V488I同时出现在3株耐药株中;(6)D116E、E266D和V488I同时出现在2株耐药菌株中;(7)D116E、E266D和V437I同时出现在2株耐药菌株和1株S-DD株中。另外,我们也发现单突变模式比如V437I和K342R也存在于耐药菌株中。结论: 1.白假丝酵母菌敏感菌株和耐药菌株ERG11基因的突变各不相同,多位点错义突变模式和单位点错义突变是导致耐药的分子基础,多位点错义突变是剂量依赖敏感株和耐药株中ERG11基因突变的主要形式,也可能是导致耐药的主要机制之一。 2.首次报导D116E、K128T、Y132H和G465S,D116E、E266D和V437I及D116E、E266D和V488I等多位点错义突变模式。
[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.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R379

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