口腔感染中假丝酵母菌的构成及耐药研究

Study on the composition of Candida spp.in oral infections and their drug resistance

目的研究口腔感染假丝酵母菌构成以及耐药情况。方法选取2016年1月-2017年6月本院诊治口腔感染假丝酵母菌患者462例,收集口腔患处样本并培养。采用显色培养基和VITEK全自动微生物分析仪鉴定菌株。采用K-B纸片法检测假丝酵母菌对临床常用的5种抗生素耐药情况,判读标准依据CLSI 2017。将CAP1-mRNA基因进行荧光定量扩增,并比较白色假丝酵母菌耐药株和敏感株的CAP1-mRNA表达水平。对耐药株ERG11基因PCR扩增,阳性产物双向测序。结果共培养出假丝酵母菌属173株,其中白色假丝酵母菌、光滑假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌和克柔假丝酵母菌分别为105、38、17、9和4株,分别占60.69%、21.97%、9.83%、5.20%和2.31%。12株白色假丝酵母菌对除两性霉素外其余四种临床常用抗真菌药物均产生不同程度耐药,光滑假丝酵母菌仅2株对氟康唑产生耐药,其他假丝酵母菌未产生耐药。12株白色假丝酵母菌中6株是多重耐药菌,2株对四种抗生素同时产生耐药性。12株白色假丝酵母菌耐药株CAP1-mRNA表达水平为9.5±2.21,敏感株表达水平为7.2±1.87,差异有统计学意义(t=2.45,P<0.05);12株菌株ERG11基因不同程度发生位点突变,共计发生同义突变11处,错义突变14处。结论白色假丝酵母菌是口腔假丝酵母菌感染中的优势菌种。白色假丝酵母菌耐药与CAP1-mRNA表达水平和ERG11基因位点突变关系密切。

Objective To study the composition and drug resistance of Candida spp.in oral infections MethodsSubjects were 462 patients who were diagnosed with and treated for an oral Candidainfection at this Hospital from January 2016 to June 2017.Samples from the oral cavity were collected and cultured.Strains were identified using chromogenic medium and the VITEK automated microbial analyzer.The resistance of Candida albicans to 5 commonly used antibiotics was determined using K-B disc diffusion,and the results were interpreted in accordance with the 2017 guidelines of the CLSI.The CAP1 gene was amplified using real-time fluorescent quantitative PCR.The ERG11 gene was amplified with PCR,and final products were sequenced bidirectionally. Results A total of 173 Candidastrains were isolated.One hundred and five（60.69%）were C.albicans,38（21.97%）were C.glabrata,17（9.83%）were C.tropicalis,9（5.20%）were C.parapsilosis,and 4（2.31%）were C.krusei.Twelve strains of C.albicans had varying degrees of resistance to all drugs except amphotericin.C.glabrata was resistant to fluconazole alone and was not resistant to other drugs.Other Candidaspecies were sensitive to 5 commonly used antibiotics.Six strains of C.albicans were multidrugresistant and 2 were resistant to 4 drugs.The level of CAP1-mRNA expression was 9.5±2.21 in resistant C.albicans and 7.2±1.87 in sensitive C.albicans.The level of CAP1-mRNA expression in resistant and sensitive C.albicans differed significantly（t=2.45,P〈0.05）.Different mutations were noted,including 11 synonymous mutations and 14 missense mutations. Conclusion C.albicansis still the predominant strain in oral Candidainfections.The drug resistance of C.albicans was closely related to the level of CAP1-mRNA expression and mutation of the ERG11 gene.