Isolation Of Candida Parapsilosis From Patients With Fungal Infection And Screening And Preliminary Verification Of Functional Genes Related To Its Growth And Metabolis

Objective: To study the clinical characteristics of deep Candida infections and Candida parapsilosis infections in Guizhou,and screen and verify the functional genes related to the growth and metabolism of Candida parapsilosis based on pretranscriptional sequencing information,laying a foundation for the prevention and treatment of Candida parapsilosis infections and the development of antifungal drugs base.Methods: 1.The strains were isolated and cultured on Sabouraud dextrose agar medium,and the strains were observed by Gram staining method and CHROMagar chromogenic medium.The strains were identified by BACTEC? FX instrument and Phoenix-100 instrument.By sequencing the ITS sequences and D1/D2 sequences of the strains,more accurate identification of bacterial species.Select some hematogenous clinical strains of Candida parapsilosis to detect their biofilm-forming capacity.2.Test the in vitro drug susceptibility of Candida with the ATB FUNGUS 3susceptibility kit.3.The transcriptome sequencing results of Candida parapsilosis of the antimicrobial peptide MAF-1A were analyzed by GO and KEGG enrichment analysis,and the key functional genes of Candida parapsilosis growth and metabolism were screened out,and the transcriptome sequencing results were preliminarily verified by q RT-PCR technology.QRT-PCR technology was used to detect the expression levels of COX15 and CDC28 genes of different biofilm-forming strains.Results: 1.A total of 1917 strains of Candida infection were isolated and identified,of which Candida albicans dominated(49.2%),followed by Candida glabrata(19.0%),Candida tropicalis(15.8%),Candida parapsilosis(8.5%),and Candida krusei(4.5%).The main causes of bloodstream Candida infections were Candida parapsilosis(37.6%),Candida albicans(27.8%)and Candida glabrata(18.8%).2.5-Fluorocytosine and amphotericin B show strong sensitivity to most Candida.In addition to the Candida parapsilosis,several other types of Candida are significantly resistant to azole drugs and are cross-resistant.Among them,Candida albicans,Candida glabrata and Candida tropicalis showed high resistance to itraconazole,with drug resistance rates of 6.9%,15.9% and 18.0%,respectively,and Candida krusei had the highest resistance to fluconazole,reaching 15.7%.3.The Candida parapsilosis complex in Guizhou area included Candida parapsilosis sensu stricto(73.0%),Candida orthopsilosis(6.1%)and Candida metapsilosis(20.9%).Different genotype strains had no significant resistance to common antifungal drugs.4.Among the 13 clinical strains of Candida parapsilosis,92.3% of the strains had biofilm formation ability,and 2(15.4%),4(30.8%)and 7(53.8%)strains had strong,general and weak biofilm formation capabilities,respectively.5.The sequencing analysis found that MAF-1A had 2187 gene expression differences in nearly 6 hours of Candida parapsilosis,and through GO and KEGG enrichment analysis,it was found that the differentially expressed genes were most significantly enriched in the oxidation-reduction process and Biosynthesis of secondary metabolites,and the functional genes COX15 and CDC28 related to mitochondrial respiratory function and cell cycle in these two processes were further screened,and the transcriptome sequencing results were verified to be feasible.The expression of COX15 and CDC28 in strains with different biofilm formation capabilities was detected by q RT-PCR,and it was found that COX15 and CDC28 were upregulated in strains with strong biofilm formation ability and general ability.Conclusions: 1.Candida albicans in Guizhou is the main bacterial species that causes deep Candida infections,but other non-albicans Candida is increasing year by year,especially Candida glabrata,Candida tropicalis and Candida parapsilosis are common infectious Candida;Bloodstream infections are mainly Candida parapsilosis,Candida albicans and Candida glabrata.The isolated Candida showed strong sensitivity to 5-flucytosine and amphotericin B,and strong resistance and crossresistance to azoles.2.The isolated Candida parapsilosis complex is mainly Candida parapsilosis sensu stricto infection,and the three genotype strains have no obvious resistance to antifungal drugs.3.The intervention effect of MAF-1A on Candida parapsilosis may mainly affect its mitochondrial respiratory function and cell cycle processes,COX15 and CDC28 may be involved in the production of cytochrome c and the regulation of cell cycle in these two processes,and even affect the production of reactive oxygen species and the stability of the cell cycle of Candida parapsilosis,and it is found that these two genes may have a certain impact on the formation of the biofilm of Candida parapsilosis.