Detection Of ERG11 Mutations In Fluconazole-resistant Candida Albicans Isolates And Application Of DNA Array In Identification

Candidiasis is becoming more common,following the widespread application ofbroad-spectrum antibiotics,immunodepressants,therapeusis of intervention and organ transplantation.And the utilization of antifungal agents promotes the process of resistance formation.More and more researchers pay their attention to the molecular mechanisms of candidal resistance to antifungal agents,especially to azoles,in order to find the way to rapid diagnosis and effective treatment of candidasis.Azoles opened a new world of treatment in candidasis,but in the same time, azole-resistant isolates are more frequently confronted with under the drug selection pressure.Candidal resistance is an arising medical problem,attracting people's notice. Identification of pathomycetes and susceptibility test play an important role in the clinical treatments and prognostic decisions.Candida albicans is the most important pathomycete of candiasis.Overseas researches on molecular mechanisms of C.albicans to azoles have been carried out. Up to now,there are 4 major hypotheses on azole-resistance mechanisms of Candida spp..The first is based on spatial configuration changes of the target enzyme 14 alpha-demethylase(Erg11p)due to mutations in the encoding gene ERG11.Erg11p is a key enzyme in the ergosterol synthesis pathway of C.albicans.Ergosterol is essential for maintaining the integrity and function of C.albicans membrane.Azoles can bind with the enzyme and block ergosterol synthesis.If one or more mutations in ERG11 result in changes in the Erg11p spatial configuration,a decrease in the affinity between the azole and protein occurs.This altered phenotype often makes isolates resistant to azole.In addition,over expression of ERG11 has been thought to increase resistance,although recent data indicate that over expression is unrelated to azole resistance in C.albicans.Amino acid substitutions have been described in 61 residues of the Erg11p due to missense mutations in ERG11 gene.Erg11p mutations Y132H, T315A,S405F,G464S,R467K and I471T have been shown to cause azole resistance.Multidrug transporters also contribute to drug resistance in Candida species. Over expression of candida drug resistance genes CDR1,CDR2 as well as the multiple drug resistance gene MDR1 can decrease the intracellular drug concentration effectively.Also,biofilms may mediate antifungal resistance.The C.albicans biofilm like that of other species is a highly heterogeneous bilayered structure,composed of cellular and noncellular elements and a matrix consisting of carbohydrate,protein and other components.Biofilms represent a niche for microorganisms where they are thought to be protected from the host immune system and antimicrobial therapies. Finally,fluconazole sequestration within intracellular vacuoles may be a novel mechanism of resistance.Multiple mechanisms of fluconazole resistance can arise in a single C.albicans isolate,and there also are chances that even a single base change in ERG11 can increase resistance to azoles.Microarray technology can provide a platform for us to identify such mutations in ERG11 and to get information of isolates' susceptibility to fluconazole,without any susceptibility test.There are different names for the microarrays,like DNA/RNA Chips,BioChips or GeneChips.The array can be defined as an ordered collection of microspots,each spot containing a single defined species of a nucleic acid.The microarray technique is based on hybridisation of nucleic acids.In this technique,sequence complementarity leads to the hybridisation between two single-stranded nucleic acid molecules,one of which is immobilised on a matrix,such as membranes,glass or silicon chips. Microarrays are fabricated either by in situ light-directed chemical synthesis or by conventional synthesis followed by immobilisation on a glass substrate.The major feature of this technique is that it allows one to perform a simultaneous analysis of a great number of DNA sequences.The microarray technology,as one of the most important technologies in post-genomics times,may be employed in diagnostics(mutation detection),gene discovery,gene expression and mapping.It is used to measure expression levels of genes in bacteria,plant,yeast,animal and human samples. Somebody detected several genes in candidal genomics and compared fluconazoleresistant and -sensitive isolates with microarray.In another study,microarray was used to identify fungal species including candida.They considered microarray as a useful technique with extremely high sensitivity and specificity.However,there was no report concerning identification of resistant isolates of C.albicans with microarray.Objectives:to investigate candida species distribution and susceptibility of candida isolates to fluconazole;to screen mutations ERG11 gene of C.albicans isolates and analyze the relationship between mutations and fluconazole resistance;to fabricate DNA microaray,identify frequent candida species and C.albicans ERG11 mutations resulting in fluconazole-resistance.Methods:Clinical isolates of candida species were collected and stored on modified SDA slants at 4℃after inoculating specimen from patients with candidiasis. CHROMagar medium is used to identify C.albicans(including C.dubliniensis),C. tropicalis,C.glabrata,and C.krusei.Germ tube formation test,saccharobiose assimilation test,and chlamydospore formation test on Corn meal-tween agar(CMA),were used to confirm the identification of C.albicans(including C.dubliniensis). Triphenyltetrazolium chloride(TZC)agar reduction test was used to confirm the identification of C.tropicalis.C.parapsilosis and other species were identified by VITEK2 system.The sequence including the 25S rDNA transposable intron of C. albicans(including C.dubliniensis)isolates was amplified by PCR to differentiate these two species and subtype C.albicans isolates.Fluconazole susceptibility was tested in vitro using microdilution and disc diffusion assays to decide sensitive isolates(S),dose-dependent sensitive isolates (S-DD)and resistant isolates(R).The microdilution test was performed referring the M27-A2 broth dilution methods protocol as recommended by the Clinical and Laboratory Standards Institute(CLSI).The results from disc diffusion assays were referred when the results of microdilution test were read,and if there was any conflict, corresponding susceptibility tests should be repeated.The final results were in accordance with those from microdilution test.Three pair of primers were designed referring C.albicans ERG11 sequence of GenBank X13296 and synthesized to amplify the ERG11 gene of fluconazole-resistant isolates,2 resistant type strains,4 sensitive type strains and several sensitive isolates of C.albicans with Pfu DNA polymerase.The 2 resistant type strains with definite ERG11 sequence were as quality control(QC).PCR products were purified with gel purification kit and then sequenced.At least two perfectly matching probes and one mismatching probe were designed according to each of the following mutations in ERG11:T541C,A1090G, C1361T,G1537A,G1547A,T1559C.One species-specific probe was designed according to each of the following six fungi' internal transcribed spacer ITS2 conserved sequence:C.albicans,C.dubliniensis,C.tropicalis,C.glabrata,C.krusei and C.parapsilosis.DNA microarray was fabricated with OmniGrid^TM100.Every probes were applied 3 times.ERG11 ORF and species-specific sequence of ITS2 in type strains C2a(C.tropicalis),Y10a(C.glabrata),C8a(C.dubliniensis),ATCC6258 (C.krusei),ATCC22019(C.parapsilosis),as well as in those sequenced,were amplified by double PCR.The products were purified with NaAc/alcohol and then incised with DNaseⅠto form fragments of about 100 bp in length.Six ERG11 oligonucleotides embracing T541C,A1090G,C1361T,G1537A,G1547A,or T1559C respectively and six species-specific oligonucleotides from ITS2 region of C.albicans, C.dubliniensis,C.tropicalis,C.glabrata,C.krusei or C.parapsilosis respectively were synthesized.These twelve 50 bp oligonucleotides and the incised PCR products were labeled with Cy3 fluorescein in a system of terminal deoxynucleotidyl transferase(TDT)and hybridized with DNA microarray.The hybridized microarray was scaned by GenePix 4000B confocal laser scanner.The values of fluorescence signal intensity were read by GenePix Pro.Results of species identification and mutation detection were decided from the values.Results:Totally,426 clinical isolates of candida species were collected.Two hundred and sisty three isolates were from VVC patients,101 isolates from sputum specimen,22 isolates from urine,16 isolates from skin or nail scraping specimen,7 isolates from blood,6 isolates from oral cavity scraping specimen,and another 11 isolates from other sites.C.albicans occupied 68.8%(293/426),C.tropicalis 12.2% (52/426),C.glabrata 10.8%(46/426),C.parapsilosis 3.3%(14/426),C.krusei 2.3% (10/426),C.dubliniensis 1.2%(5/426),and others 1.4%(6/426).Sensitivity rate of candida isolates to fluconazole is 84.5%(360/426),dose dependent sensitivity rate is 7.0%(30/426),and resistance rate is 8.5%(36/426). Sensitivity rate of C.albicans isolates to fluconazole is 89.4%(262/293),dose dependent sensitivity rate is 5.5%(16/293),and resistance rate is 5.1%(15/293). Sensitivity rate of C.tropicalis isolates to fluconazole is 92.3%(48/52),dose dependent sensitivity rate is 5.8%(3/52),and resistance rate is 1.9%(1/52). Sensitivity rate of C.glabrata isolates to fluconazole is 56.5%(26/46),dose dependent sensitivity rate is 23.9%(11/46),and resistance rate is 19.6%(9/46).All of the 15 resistant isolates of C.albicans are genotype A.ERG11 of those 15 resistant isolates,8 sensitive isolates,and 6 type strains of C. albicans was amplified and sequenced.Thirty seven mutations were detected,of which 19 were missense mutation,and the other 18 were silence mutation.Eight missense mutations existed in the resistant strains or isolates,while 13 missense mutations existed in the sensitive.ERG11 sequence of resistant type strains by sequencing was same with what had been reported.In each of 14 resistant isolates, mutation G487T and mutation T916C were detected simultaneously without any other mutation in ERG11 sequence.In the other resistant GZ04,4 missense mutations T495A,A530C,T541C,and T1493A were detected.In the sequences of sensitive type strains,homozygous T495A,A504C,G820T and A945C and heterozygous T495A/C, T566G/T,G630T/G,G635C/G,C1271A/C and G1289T/G were found.No missense mutation was detected in the sensitive isolates C261,0461 or 2855.Homozygous T495A,A530C,G640A,G820C and A945C and heterozygous A945C/A and G1609A/G were found in the other 5 sensitive isolates.Thirty four strains and 12 oligonucleotides were identified by DNA microarray hybridization and correct results were obtained in species identification and mutation detection.Both sensitivity and specificity are 100%.Conclusions:(1),C.albicans is still the major cause of candidiasis.Both its proportion in composition of Candida isolates and the sensitivity of Candida spp.to fluconazole are similar with the reported data.(2),the mutations G487T(A114S)and T916C(Y257H)might participate in the formation of resistance to fluconazole,while the following mutations A504C (K119N),G820T(D225Y),G820C(D225H)or G640A(E165K)cannot.(3),it is a reliable method to identify candida species and ERG11 mutations by DNA microarray hybridization.