| BackgroundInvasive candidiasis is the most common invasive fungal infection,with high morbidity,high mortality,and high diagnosis and treatment costs.Candida albicans is still the most frequent etiologic agent in invasive candidiasis patients,but the incidence of non-albicans Candida is gradually increasing in recent years and these species have the potential to cause outbreaks.Currently,available systemic antifungal drugs mainly include triazoles,polyenes and echinocandins,among which echinocandins are the leading choices for invasive candidiasis.With the extensive use of antifungal drugs in clinic,drug resistance of Candida is increasing.Meanwhile,new etiologic agents,such as the super fungus Candida auris,are emerging.The above antifungal drugs will unable to meet clinical needs and mortality of invasive candidiasis remains high.Part one: Collection and species identification of Candida isolates in patients with invasive candidiasisObjectiveTo analyze the epidemiological data of patients with invasive candidiasis and grasp the species distribution characteristics of Candida isolates.MethodsThe Candida strains detected from patients with invasive candidiasis who visited Shanghai Changzheng Hospital from 2015 to 2019 were collected,and the clinical information of the patients was systematically sorted out,including gender,age,department and main diagnosis.DNA samples from experimental strains were PCR amplified using two different fungal universal primers,ITS1/ITS4 and D1/D2.After sequencing,the Candida strains could be identified to the species level.ResultsA total of 107 Candida strains were included in this study,derived from 107 patients.There were 31 strains of C.albicans,34 strains of Candida parapsilosis complex(30 strains of C.parapsilosis,3 strains of Candida orthopsilosis,1 strain of Candida metapsilosis),17 strains of Candida glabrata,14 strains of Candida tropicalis,5 strains of Candida guilliermondii,4 strains of Candida haemulonii,and 2 strains of Candida krusei.57 strains were derived from blood samples,and 50 strains were derived from other sterile specimens such as catheters,puncture fluid,dialysate and ascites.There were 63 males and 44 females.The age distribution ranged from 18 to 87 years old,with an average of 59.03±14.61 years old,and 62 patients were ≥60 years old.The top three source departments were 28 cases in emergency department,18 cases in nephrology department,and 14 cases in general surgery department.The main clinical diagnoses were acute and critical disease,tumor,operation,kidney disease and other autoimmune diseases.Based on the results of molecular biology sequencing,the strains were preliminarily identified by conventional means,and 12.15%were identified with deviations.ConclusionThe Candida strains collected in this study mainly include seven species,including C.albicans,C.parapsilosis complex,C.glabrata,C.tropicalis,C.guilliermondii,C.haemulonii and C.krusei.Although C.albicans is still the most important type of pathogen,the proportion of non-albicans Candida is as high as 71.03%.C.parapsilosis complex accounts for the highest proportion of blood samples,and C.albicans accounts for the highest proportion of other sterile samples.It is more common in elderly patients and people with impaired autoimmunity,and the proportion of male patients is higher than that of female patients.Compared with traditional morphological and biochemical detection methods,molecular biology technique has the advantages of high sensitivity,high specificity and short detection time.Part two: In vitro antifungal susceptibility testObjectiveTo master the in vitro susceptibility of Candida isolates to different antifungal drugs and provide reference for the clinical application of antifungal drugs.MethodsThe in vitro sensitivity of the experimental strains to fluconazole,itraconazole,voriconazole,posaconazole,caspofungin,micafungin,anidulafungin,amphotericin B and5-fluorocytosine were detected by the M27-A3 yeast dilution method recommended by the American Clinical and Laboratory Standards Institute.ResultsThe susceptibility rates of C.albicans to the four triazoles were all over 80%,96.77%of the strains were sensitive to the three echinocandins,and all strains were sensitive to amphotericin B and 5-flucytosine.No drug-resistant strains were found in C.parapsilosis and C.metapsilosis,and two strains of C.orthopsilosis were cross-resistant to fluconazole,voriconazole and capofungin.Meanwhile,the MIC of C.parapsilosis complex was lower to triazoles,but higher to echinocenins.The drug resistance rates of C.glabrata and C.tropicalis to the four triazoles were higher.All strains of C.glabrata were sensitive to echinocandins and amphotericin B.One strain of C.tropicalis was resistant to micafungin,and all strains of C.tropicalis were sensitive to amphotericin B.One strain of C.glabrata and one strain of C.tropicalis were resistant to 5-fluorocytosine.Except for two strains resistant to 5-fluorocytosine,all strains of C.guilliermondii were sensitive to other drugs,with lower MIC to voriconazole and higher MIC to echinocandins.All strains of C.haemulonii were sensitive to all three echinocinins,and drug-resistant strains appeared to other drugs.All strains of C.krusei were naturally resistant to fluconazole,and no resistance to other drugs was found.ConclusionAlthough C.albicans has cross-resistance to triazoles,there is no large-scale drug resistance,and it has satisfactory sensitivity to echinocandins.Triazoles can be the first choice for empiric treatment of C.parapsilosis and C.metapsilosis invasive infection,and fluconazole,voriconazole and caspofungin should be used cautiously for C.orthopsilosis invasive infection.Echinocandins are the first-line regimen for the treatment of C.glabrata and C.tropicalis invasive infection.Voriconazole is the first choice for the treatment of C.guilliermondii invasive infection.Echinocandins can be the first choice for the treatment of C.haemulonii invasive infection.Except for fluconazole,all other drugs can be considered for the treatment of C.krusei invasive infection.Except for C.haemulonii,the sensitivity of amphotericin B and 5-fluorocytosine to Candida strains are satisfactory.Part three: Transcriptomic study of fluconazole-resistant Candida tropicalisObjectiveTo study the transcriptional regulation of fluconazole-resistant C.tropicalis when exposed to fluconazole using transcriptome sequencing technology,analyze the genes and pathways related to the drug,and try to explore the drug resistance mechanism.MethodsThe drug-resistant C.tropicalis strain was treated with fluconazole.The transcripts were sequenced using RNA-seq,the sequencing results were compared to the reference genome after quality control,and then the function of the transcript gene were analyzed.Reference databases used included NR,KEGG,Egg NOG,Swiss Prot,CAZy,MERPOS,PHI,and Fungal P450 Enzyme Database.The differentially expressed genes were analyzed,and the differentially expressed pathways and functions were enriched.Protein structures were predicted using Alpha Fold and homologous structures were searched using Foldseek.ResultsC.tropicalis MYA-3404 was selected as the reference genome(Gen Bank Accession:GCF＿000006335.3).The alignment rates ranged from 95.25% to 95.67%,and a total of 6182 expressed genes were detected.More than 99% of the reads aligning to the CDS region were found in six samples.A total of 188 genes were predicted to be peptidases,226 genes encoded carbohydrate activity-related enzymes,more than 1000 protein-coding genes were predicted to be associated with pathogenicity of C.tropicalis,and 198 potential CYP450 were identified.Using TPM as a quantitative indicator,a total of 160 differentially expressed genes were screened,of which 66 were up-regulated and 94 were down-regulated.Pathway enrichment was mainly concentrated in sulfur metabolism,pyruvate metabolism,glycolysis and gluconeogenesis,amino acid metabolism,unsaturated fatty acid synthesis and mitosis.The functions of the differential genes were largely enriched in membrane intrinsic components,transport and redox.Structural annotation and functional prediction of CTRG＿03267 was ornithine hydroxylase,which was involved in the alteration of membrane composition.ConclusionIn this research,one fluconazole-resistant C.tropicalis strain is studied,and RNA-seq is used to analyze the transcriptional regulation of this strain under drug stress when fluconazole is added.It is speculated that the drug resistance of this strain mainly comes from the overexpression regulation of transporters and the change of membrane components,as well as the tightening of amino acid synthesis and energy metabolism to reduce the growth rate.At the same time,the structure prediction and functional annotation of a highly expressed gene without functional annotation are performed,and a structure prediction model with high confidence is obtained.The product of this gene is predicted to be an ornithine hydroxylase,and the regulation of membrane composition is achieved by upregulating the expression of this enzyme. |
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