Molecular Mechanisms On Itraconazole Resistance Of Candida Krusei Clinical Isolates

Objective: To study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei.Methods: 1.Clinical strains The isolates of C.krusei included in the present study were from a collection of clinical isolates recovered from Tianjin First Central Hospital.They were identified using Candida chromogenic medium,Vitek2 YBC identification cards and mass spectrometer.2.Antifungal susceptibility testing Reference antifungal susceptibility testing of the study isolates was performed by ATB FUNGUS 3 according to the manufacturer's instructions,and visually read the minimal inhibitory concentration(MIC)value.Testing on the ATB FUNGUS 3 was performed simultaneously with CLSI BMD method.The susceptibility profiles to itraconazole of the C.krusei isolates were as follows: susceptible(S)whenever MIC 0.125?g/ml,susceptible dose dependent(SDD)whenever MIC = 0.25 to 0.5 ?g/ ml and resistant(R)whenever MIC 1.0?g/ml.Visual readings were performed after 24 h of incubation.Quality control was ensured by testing the NCCLS-recommended strains C.krusei ATCC 6258 and C.parapsilosis ATCC 22019.3.Amplifying and sequencing analysis of ERG11 gene Total genomic DNA was extracted using DNA extraction kit.The 14?-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction(PCR),and their nucleotide sequences were determined to detect point mutations.4.ABC1,ABC2 and ERG11 genes expression analysis Total RNA was extracted using Trizol reagent.ERG11 and efflux transporters(ABC1 and ABC2)genes were determined by quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)for their expression in itraconazole-resistant(R),itraconazole-susceptible dose dependent(SDD)anditraconazole-susceptible(S)C.krusei at the m RNA level.Results: 1.These isolates were recovered from various body sites of 16 different patients.Among the isolates studied,there were 5 itraconazole-resistant(R)strains,8 itraconazole-susceptible dose dependent(SDD),and 3 itraconazole-susceptible(S)isolates.2.We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates,including 6 synonymous mutations and 1 missense mutation(C44T).However,the missense mutation was found in the three groups.3.The m RNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones(P=0.015 and P=0.002 respectively).ABC2 gene m RNA levels in itraconazole-resistant group was significantly higher than the other two groups,and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole(P=0.007 in SDD compared with S,P=0.016 in SDD with R,and P<0.001 in S with R respectively).While ABC1 gene presented lower expression in itraconazole resistant strains.However,the m RNA levels of ERG11,ABC1 and ABC2 in a C.krusei(CK10)resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates.Conclusions: 1.There are ERG11 gene polymorphisms in clinical isolates of Candida krusei.ERG11 gene mutations were not found to be involved in the development of itraconazole resistance in Candida krusei.2.ERG11 and ABC2 upregulation might be responsible for the acquired itraconazole resistance of these clinical isolates.