| Aspergillus spp.includes a variety of species that can cause major health problems,such as A.fumigatus,A.flavus,A.niger,and A.terreus.Infections caused by Aspergillus,collectively known as aspergillosis,are more common in immunocompromised people.In recent years,the incidence of aspergillosis has increased year by year,second only to candidiasis,and has ranked second in deep fungal infections and first in filamentous fungal infections.Due to the few types of antifungal drugs,the greater toxicity and side effects,and the increasingly serious drug resistance of pathogenic bacteria,the mortality rate of the disease is high.Among them,A.fumigatus is the most important opportunistic pathogenic fungus in clinical practice,accounting for about 90% of all Aspergillosis.Due to the widespread use of clinical antifungal drugs and agricultural antifungals,drug-resistant strains of A.fumigatus continue to emerge.Increasing rates of drug resistance make treatment particularly difficult.At present,many studies on fungal drug resistance mechanisms have been reported,such as ergosterol synthesis pathway,drug efflux,and biofilm changes.Studies have shown that Aspergillus drug resistance is a complex multi-gene regulation process,and there are still many regulatory mechanisms that remain unclear.Understanding the drug resistance of Aspergillus and exploring the regulatory mechanism of drug resistance-related genes can provide a certain reference for the development of new drugs and clinical treatment.In this study,the susceptibility of 75 strains of Aspergillus isolated from the environment and clinical to commonly used antifungal drugs was analyzed.The 75 strains of Aspergillus were identified by morphology and molecular biology,including39 strains of A.fumigatus,27 strains of A.niger,8 strains of A.flavus and 1 strain of A.terreus.The drug susceptibility test results showed that the resistance rate of A.fumigatus group posaconazole（POS）was 5.1%,the resistance rate to itraconazole（ICZ）and voriconazole（VCZ）was 7.7%,and the resistance rate to amphoteric mold was 7.7%.The resistance rate of Am B（Am B）was 10.2%;the resistance rate of A.niger to ICZ was 18.5%,the resistance rate to VCZ was 25.9%,the resistance rate to POS was 3.7%,and sensitive to Am B;A.flavus was 12.5% resistant to ICZ and Am B,37.5% resistant to POS,and sensitive to VCZ.It is suggested that A.fumigatus has the highest resistance to Am B,followed by ICZ;A.niger has the highest resistance to VCZ,followed by ICZ,and is sensitive to Am B;A.flavus has higher resistance to ICZ,POS and Am B,are sensitive to VCZ.The results provide a certain reference for clinical treatment and drug selection.Three A.fumigatus isolates（h11,h12,and h58,respectively）were found to be less sensitive to azoles than A.fumigatus IFM40808.In order to explore the regulatory mechanism of drug resistance,the known drug resistance genes related to ergosterol synthesis pathway and efflux pump related drug resistance genes were sequenced and analyzed by q PCR to detect m RNA expression levels and gene mutations.The results showed that compared with the susceptible strain,the cyp51 A,cyp51B and hmg1 genes of strain h11 had no mutation,but the expression of cpr1 and cyp51 A genes was up-regulated,indicating that the reason for its drug resistance was related to the expression of azole drug target genes in the ergosterol synthesis pathway.related upwards.The mutation of the cyp51 A gene of strain h12 resulted in the 98 th amino acid mutation（L98H）,but the expression level of drug resistance-related genes was not significantly up-regulated,indicating that the drug resistance was caused by the amino acid mutation of the target gene of azole drugs.The amino acid sequence of the resistance-related genes of strain h58 was not mutated,and the m RNA expression level was not significantly up-regulated,indicating that the resistance mechanism of this strain is different from that reported so far.In order to deeply explore the regulation mechanism of the drug resistance of A.fumigatus h58,the fungal cell wall integrity was analyzed.The ergosterol content of the strain was detected,and the results showed that there was no significant difference compared with the susceptible strain,indicating that its drug-resistant phenotype had nothing to do with ergosterol synthesis.The results of itraconazole efflux experiments showed that compared with the susceptible strain,the drug content in the culture medium of this strain was significantly increased,indicating that the increased drug efflux might be the reason for its drug resistance.In order to find the genes related to the regulation of drug resistance,the transcriptome sequencing analysis of the sensitive strain and the resistant strain h58 was performed,one group was the drug-free group,and the other group was the itraconazole-treated group.The results showed that compared with the susceptible strains,a total of 876 genes were differentially expressed in the resistant strains in the drug-free group,of which 480 were up-regulated and 369 were down-regulated.A total of 458 genes were up-regulated and 273 genes were down-regulated in the itraconazole-resistant strains.The differentially expressed genes were analyzed,and it was found that the expression of efflux transporter-encoding genes was up-regulated,and the redox-related genes were significantly enriched,among which the expression of glia encoding the gliotoxin efflux transporter and the superoxide dismutase gene sod2 were the most altered.To explore the relationship between these two genes and drug resistance,gene-directed knockout of glia and sod2 in drug-resistant strains was performed.The results showed that the deletion of the gene glia would affect the content of gliotoxin produced by the strain but not its sensitivity to azole drugs.Increased sensitivity to posaconazole and oxidative stress following deletion of the gene sod2.In conclusion,this study took 75 strains of Aspergillus isolated from the environment and clinic as the research objects to analyze the drug resistance status.Among them,A.fumigatus had the highest resistance to Am B,and had different degrees of resistance to azoles;A.niger was sensitive to Am B;A.flavus was sensitive to VCZ.The analysis of ergosterol-related pathways and efflux pump-related resistance genes of three drug-resistant A.fumigatus strains showed that the resistance of two strains was related to the mutation and up-regulation of azole drug target genes in the ergosterol synthesis pathway,there is 1 strain different from the resistance mechanism reported so far.Genes that may be associated with drug resistance were screened out based on transcriptome sequencing analysis and verified by gene knockout.The results showed that the deletion of the gliatoxin efflux transporter gene glia did not affect drug sensitivity;the deletion of the manganese superoxide dismutase gene sod2 increased the sensitivity to posaconazole.The results of this study can provide a certain reference for the selection of clinical treatment drugs and the revealing of the drug resistance mechanisms of A.fumigatus. |
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