念珠菌ERG3基因突变对抗真菌药物耐药的作用

发布时间：2018-12-13 19:19

【摘要】：背景近年来，侵袭性真菌感染发病率不断上升，以念珠菌感染为主，其中白念珠菌为主。但由于预防性使用抗真菌药物，非白念珠菌尤其是对氟康唑天然耐药的克柔念珠菌感染出现上升趋势，但白念珠菌感染仍占优势。随着白念珠菌和克柔念珠菌对唑类抗真菌药物的耐药现象越来越严重，深入研究其耐药机制迫在眉睫。目的调查本院住院患者侵袭性真菌感染的病原学特征及相关危险因素。了解白念珠菌和克柔念珠菌对常用抗真菌药物的敏感性，对白念珠菌耐药菌株和敏感菌株ERG3基因扩增、测序、生物信息学分析，明确白念珠菌ERG3基因突变与抗真菌药物耐药之间的关系。方法收集2006-2010年山西医科大学第二临床医学院452例住院患者无菌体液标本，使用沙氏培养基、玉米吐温80琼脂培养基、CHROMagar念珠菌显色培养基和API 20C AUX鉴定系统进行念珠菌的分离和鉴定。从中分别选取35株白念珠菌和20株克柔念珠菌进行体外药物敏感性试验，确立耐药菌株和敏感菌株。对白念珠菌ERG3基因进行PCR扩增、测序，最后应用BLAST软件进行比对分析。结果 (1)检出的163株真菌中，，白念珠菌54.61%，非白念珠菌42.94%，霉菌2.45%。其中非白念珠菌从高到低依次为克柔念珠菌(16.57%)、光滑念珠菌(14.72%)、热带念珠菌(4.91%)、近平滑念珠菌(4.29%)和葡萄牙念珠菌(2.45%)；Logistic多因素回归分析结果显示：性别、病程、体温、预防性使用抗真菌药物、免疫抑制剂、有创伤的检查和治疗、患者预后和地域因素是住院患者无菌体液标本中侵袭性真菌感染的独立危险因素。 (2)从中选取的35株白念珠菌体外药物敏感性试验结果显示：白念珠菌对5-氟胞嘧啶和两性霉素B敏感性为100%，对伊曲康唑、伏立康唑和氟康唑有不同程度的耐药，耐药率分别是17.14%、11.43%、8.57%，而且存在交叉耐药和多重耐药情况；20株克柔念珠菌体外药物敏感性试验结果显示：克柔念珠菌对伏立康唑和两性霉素B有较高的敏感性，5-氟胞嘧啶、氟康唑和伊曲康唑有不同程度的耐药，耐药率分别是20.00%、50.00%、45.00%，也有交叉耐药和多重耐药现象。 (3)本研究显示白念珠菌临床耐药菌株和敏感菌株的ERG3基因均存在突变位点，共检出9个点突变，其中耐药菌株A17号同时存在1个同义突变和4个错义突变。A17号菌株同义突变位点为1080bp。突变前碱基所在的密码子为GGC，所编码的氨基酸为甘氨酸，未引起氨基酸的改变。4个错义突变位点分别为I340L、V374G、P902L、V1076A，突变发生氨基酸置换。第100位碱基由A突变为C，导致第340位氨基酸由异亮氨酸转变为亮氨酸；第134位碱基由T突变为G，导致第374位氨基酸由缬氨酸转变为甘氨酸，第662位碱基由C突变为T，导致第902位氨基酸由脯氨酸转变为亮氨酸；第836位碱基由T突变为C，导致第1076位氨基酸由缬氨酸转变为丙氨酸，这4个点突变在其它文献中尚未见报道，是本实验新发现的突变位点。其余菌株均为同义突变，突变位点分别是602bp、795bp、969bp、1080bp、1110bp，未引起氨基酸改变。结论 (1)白念珠菌感染仍占优势，但非白念珠菌感染明显上升，尤其是对氟康唑天然耐药的克柔念珠菌，临床医师应重视发生的这一流行病学变化。 (2)体外药敏试验结果提示：白念珠菌对唑类药物有不同程度的耐药，并且对伊曲康唑的耐药率高于氟康唑，但大多数菌株是敏感菌株，所以唑类抗真菌药物仍然可以用于临床上白念珠菌感染的治疗。 (3)白念珠菌部分耐药菌株存在麦角甾醇合成途径中酶编码基因ERG3的错义突变，并且为多位点突变，但突变的数目和分布模式没有明显的规律性，所以ERG3基因突变在耐药机制中的作用尚需进一步证实。实验中发生的错义突变是单独突变还是同时突变才可以导致耐药，也需要进一步研究。
[Abstract]:Background In recent years, the incidence of invasive fungal infection has been increasing, and the infection of candidiasis is the main, of which Candida albicans is the main.. However, as a result of the upward trend in the prophylactic use of antifungal drugs, non-Candida albicans, especially in the case of a natural drug-resistant gram of Candida, the infection of Candida albicans is still dominant With the increasing of the resistance of Candida albicans and C. g. C. to the anti-fungal drugs, it is urgent to study the mechanism of drug resistance.. Objective To investigate the etiological characteristics and related risk of invasive fungal infection in the hospital of our hospital Factors: To understand the sensitivity of Candida albicans and C. canalis to the common antifungal drugs, the amplification, sequencing and bioinformatics analysis of the drug-resistant strains of Candida albicans and the sensitive strain ERG3, and to clarify the relationship between the ERG3 gene mutation of the Candida albicans and the drug resistance of the antifungal drugs relationship Methods: 452 cases of sterile body fluid from the second clinical medical school of Shanxi Medical University in 2006-2010 were collected, and the isolation of Candida was carried out using the medium of Shahlet, the agar medium of Tween 80, the chromogenic medium of CHROMagar and the API 20C AUX identification system. and respectively selecting 35 strains of Candida albicans and 20 strains of Candida albicans to carry out in-vitro drug sensitivity test, and establishing a drug-resistant strain and a sensitivity. and performing PCR amplification and sequencing on the candida albicans ERG3 gene, and finally using the BLAST software to perform PCR amplification, sequencing and final application of the BLAST software to perform PCR amplification, sequencing, p-point Results (1) Among the 163 fungi detected, the Candida albicans was 54. 61%, and the non-candida albicans was 42. 94%. 2.45% of Candida, of which Candida albicans (16.57%), Candida glabrata (14. 72%), Candida tropicalis (4.91%), Candida glabrata (4.29%) and Candida utilis (2.45%) were in order from high to low, and the results of logistic multi-factor regression analysis showed that gender, The course of course, body temperature, preventive use of antifungal drugs, immunosuppressants, and trauma and treatment, the patient's prognosis and regional factors are the only one of the invasive fungal infections in the sterile body fluid specimen of the patient. The results of the in vitro drug sensitivity test of 35 strains of Candida albicans in vitro showed that the sensitivity of Candida albicans to 5-fluorophonate and amphotericin B was 100%, and the drug-resistance rate was 17.14%, respectively. 11. 43%, 8. 57%, and there were cross-resistance and multiple drug-resistance. The results of the in vitro drug sensitivity test of 20 strains of Candida utilis showed that the sensitivity, 5-fluorophonate, fluconazole and Idaikang were more sensitive to the test results of the in vitro drug sensitivity test of 20 strains of Candida utilis. In the same degree, the resistance rate was 20. 00%, 50. 00%, 45. 00%, respectively. (3) There was a mutation site in the ERG3 gene of the clinical drug-resistant strain of Candida albicans and the sensitive strain, 9 point mutations were detected, among which there were 1 synonymous mutation at the same time of the drug-resistant strain A17. and 4 missense mutations. It is 1080bp. The codon of the pre-mutation base is GGC, the encoded amino acid is glycine, and the change of amino acid is not caused. The four missense mutation sites are I340L, V374G, P902L, V1076A, and mutation. The amino acid substitution occurs. The 100-th base is changed from A to C, resulting in the transformation of the 340-th amino acid from isoleucine to leucine; the 134-th base is changed from T to G, leading to the conversion of the amino acid at position 374 to glycine, and the second base The mutation of the base from C to T, leading to the transformation of the amino acid at position 902 into leucine by the proline; the base of the 836 is changed from T to C, resulting in the 1076 amino acid being converted to alanine by the amino acid, the four point mutations have not been reported in other documents, which is the new experiment The other strains were the same. The mutation sites were 602bp, 795bp, 969bp, 1080bp and 1110bp, respectively. cause Conclusion (1) The infection of Candida albicans is still dominant, but the infection of non-Candida albicans is obviously increased, especially in the case of the natural drug-resistant Candida, the clinician should attach importance to the occurrence. This epidemiological change. (2) The results of in vitro drug sensitivity test suggest that Candida albicans has a different degree of resistance to the drug, and the drug resistance rate of the drug is higher than that of Flukang, but most of the strains are sensitive strains, so the anti-fungal drugs can still be used for clinical application. The treatment of Candida albicans infection. (3) the missense mutation of the enzyme-encoding gene ERG3 in the part of the drug-resistant strain of the Candida albicans, and the mutation of the multi-position point, but the number and the distribution pattern of the mutation have no obvious regularity, so the gene mutation of the ERG3 is in the drug-resistant mechanism. The role in the experiment still needs to be further confirmed. The missense mutation that occurs in the experiment is a single mutation or a simultaneous mutation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756

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