Resistance Analysis Of Clinical Isolates Of Fungi And Research Into The ERG11Mutations In Azole-resistant Candida Albicans In Anhui Province

ObjectiveTo investigate the species and drug resistance of the clinical fungal infections from2008to2012in Anhui Province, and to provide the evidence for clinical reasonable use of antifungaldrugs.To investigate the mutations of ERG11gene and its relationship with azole-resistant strainsof Candida albicans isolated from the clinical specimens in Anhui Province.Materials and MethodsIsolatesA total of non-duplicate464strains of S. marcescens were collected from various clinicalspecimens from40hospitals in different areas of Anhui, China from2008to2012. Qualitycontrol was ensured by Candida parapsilosis ATCC22019for susceptibility testing. This qualitycontrol strain is stored in Anhui Center for Surveillance of Bacterial Resistance.Method1.464strains selected from40hospitals of Anhui were cultured and isolated on Sabouraud’smedium. CHROMagar Candida was used for identification. The MICs of isolates weredetermined by the M27-A2broth microdilution method according to the criteriarecommended by CLSI2002.2. All the resistant isolates and three susceptible isolates selected randomly were culturedand isolated on Sabouraud’s medium. Total genomic DNA of these isolates was extractedusing the yeast DNA kit.3. Amplification of ERG11gene was carried out using the primers. All purified PCRproducts were directly sequenced by the dideoxy chain termination method. Sequencealignment was compared with the GenBank nucleotide database using the BLASTprogram (GenBank accession no. X13296).Results1. Among the selected464strains, most of the fungi were Candida albicans (69.0%), and the next were Candida glabrata (16.8%). The isolated fungi were highly sensitive toamphotericin B and5-fluorocytosine, but lowly sensitive to itraconazol and fluconazole.2. Each PCR product of all resistant isolates and3sensitive isolates presented successfully as aclear band in ethidium bromide-stained agarose gel. The size of each PCR fragment wasconsistent with the expected which should be1641bp in length.3. From the analysis of the sequencing results, we found34different base mutations,including14missense mutations.Conclusion1. The selected fungi had different levels of drug resistance in Anhui, so the surveillance offungal resistance should be strengthened, and clinical doctors should use azole antifungalagents rationally in order to improve clinical cure rate.2. The research found ERG11gene of the azole-resistant Candida albicans had manymissense mutations, and many of them were multiple point mutations, which may beconnected with drug-resistance in Candida albicans. T123I, Y334S, G346A, V452D andL480F have not been reported at home and abroad.