Pathogeny And Epidemiology Research Of Candida Albicans Isolated From Clinical Strains

Objectives This article was designed to study the distribution,identification,drug sensitivity,toxicity and homology of Candida albicans isolated from clinical speciments.This study can provide clues to explore the pathogenesis of Candida albicans and is of great significance to its epidemiology detection,prevention and treatment.Methods 1.Bacterial strains To collect Candida albicans isolated from aseptic parts of the clinical microbe laboratory of the Affiliated Union Hospital of Fujian Medical University in 2011-2016.The quality control strains are Candida albicans ATCC90028 and Candida albicans ATCC22019,which are preserved and provided by the microbiology laboratory of Fujian Medical University Affiliated Union Hospital.2.Comparison of three identification methods Candida albicans collected by the above methods were identified by CHROMagar,MALDI-TOF MS and ITS sequencing methods.3.In vitro susceptibility testing Antifungal susceptibility testing was performed using a broth microdilution method.The MIC was determined according to the broth microdilution antifungal drug sensitivity test(M27-A3)reference method approved by the National Clinical Laboratory Standardization Committee(CLSI)in 2008.4.Virulence phenotype analysis The activity of secreted hydrolytic enzymes including hemolysins,secreted aspartyl proteinases,phospholipases were determined on plates containing specific enzyme substrate(sheep blood plate,bovine serum albumin plate,yolk agar plate)by observing the formation of hemolytic rings and precipitated rings.Analysis the correlation of virulence phenotypic characteristics and compare the virulence phenotypic characteristics of Candida albicans between different strains and infection sites.5.Homology analysis Homology analysis was performed using multiple site typing(MLST),and seven housekeeping genes of Candida albicans were amplified by PCR.The amplified products were sequenced and the sequencing results were submitted to https://pubmlst.org/calbicans/.The homology of the strains was analyzed by e BURST V3 and Mega 4 software and plotted;homology analysis and mapping were performed using MSP cluster analysis and PCA analysis of MALDI-TOF MS.6.white-opaque switching MTL genotyping was performed by PCR amplification.White-opaque switching was performed using 25°C,5% CO2 Lee’s Glc NAc solid medium and the frequency of transition was determined.The activity of secreted hydrolytic enzymes of Candida albicans before and after transformation were determined on plates containing specific enzyme substrate(sheep blood plate,bovine serum albumin plate,yolk agar plate)by observing the formation of hemolytic rings and precipitated rings.7.Statistical analysis The experimental results were analyzed by SPSS20.0 software.The data were descriptively statistics with mean,standard deviation(SD),frequency,and percentage,median,etc.The data were analyzed by t-test,Chi-square test or Fisher probabilities.A two-sided test P<0.05 was considered statistically significant.Results 1.Bacterial strains A total of 173 Candida albicans strains were collected from the microbiological laboratory.In age distribution,most of them were 60-79 years old.In the sample type distribution,most of them were blood and drainage fluid specimens.Most of the strains were isolated from intensive care unit.2.Comparison of three identification methods The CHROMagar showed that 173 strains were all Candida albicans.MALDI-TOF MS showed that only 125 strains of Candida albicans were identified as Candida albicans.ITS sequencing results showed that only 125 strains were identified as Candida albicans.3.In vitro susceptibility testing 125 strains of Candida albicans were highly sensitive to fluconazole,itraconazole,voriconazole,amphotericin B,and flucytosine.Only 1.6% strains were found to be resistant to flucytosine.4.Virulence phenotype analysis In this study,100% of Candida albicans were positive for hemolysins,the mean value was 0.56±0.06,65.6% of Candida albicans were positive for protease,the mean value was 0.71±0.25,85.6% of Candida albicans were positive for phospholipase,the mean value was 0.81±0.12.There was no correlation between the three virulence phenotypes.There was no significant difference in extracellular hydrolase activity between strains from different specimens(P<0.05).5.Homology analysis For the 125 clinical isolates,61 STs were defined by MLST.According to the e BURST analysis,STs were divided into 6 groups and 6 single cases.Using the e BURST V3 and MEGA4 software respectively for homology analysis,it was found that the genetic relationship between the strains differed greatly.MSP cluster analysis and PCA analysis of the MALDI-TOF MS showed that the genetic relationship between the strains was significantly different.6.white-opaque switching Among the 125 strains of Candida albicans,5 strains were identified as MTL homozygous;125 strains were of white phenotype;26 strains of 125 strains successfully performed white-to-opaque switching,the switching rate of each strain was different,the highest was 8%.Opaque cells have higher Sap activity than white.Conclusion Among the three identification methods,the accuracy of the CHROMagar is the lowest,but it is still widely used in clinical laboratories because of its rapidity,simplicity,and low cost;MALDI-TOF MS is worth promoting in clinical laboratories for its high accuracy,less time-consuming and low cost.Clinical isolates of Candida albicans are highly sensitive to common antifungal drugs,and the virulence is closely related to geographic location.Homology analysis showed that the genetic relationship between strains differed greatly.In this study,the white-opaque switching rate was low,and the Sap activity was increased after white cells were transformed into opaque cells.