Analysis Of Relationship Between MSH2 Gene Mutation And Fluconazole Resistance In Candida Glabrata

PART ? Analysis of mutations in MSH2 gene of fluconazole-resistant Candida glabrataObjective To analyze the resistance rate of 157 C.glabrata clinical strains to five antifungal drugs,and to explore the relationship between MSH2 gene mutation and fluconazole resistance in C.glabrata clinical strains.Methods A total of 157 C.glabrata strains from clinical specimens were collected from the Department of Clinical Laboratory of the First Affiliated Hospital of Anhui Medical University from July 2017 to July 2018.The resistance to five antifungal drugs was statistically analyzed.Fluconazole-resistant(n=19),dose-dependent sensitive(S-DD)(n=15)and susceptible isolates(n=23)were randomly collected from the above 157 strains,and verified the drug sensitivity by microdilution method.Using Sanger method sequenced the MSH2 gene,and the sequencing results were compared with the published sequences in Gen Bank,and analyze of the frequency of mutations and related mutation sites in MSH2 gene.Results Of the 157 C.glabrata strains,27.4%,22.9%,5.7%,2.5% were resistance rate to itraconazole,fluconazole,voriconazole and amphotericin B,respectively.All strains were sensitive to 5-fluorocytosine.Compared with susceptible isolates(n=23),the mutation frequency of MSH2 gene in fluconazole-resistant isolates(n=19)and S-DDisolates(n=15)was significantly increased,and the difference was statistically significant(P<0.0167).The three mutation sites of G622A/A2669 T and G715 T were detected in MSH2 gene,corresponding to three amino acid substitution sites of P208S/N890 I and V239L;and P208S/N890 I only appeared in drug-resistant strains.Conclusion 157 C.glabrata clinical strains have higher resistance to azoles than amphotericin B and 5-fluorocytosine;the mutation of MSH2 gene of C.glabrata has a certain relationship with fluconazole resistance.PART ? Analysis of m RNA level of MSH2 Gene in Candida glabrataObjective To explore the relationship between m RNA level of MSH2 gene of C.glabrata clinical strains and fluconazole resistance,and further explore the relationship between MSH2 gene mutation and fluconazole resistance.Methods 19 strains of fluconazole-resistant C.glabrata strains and 23 fluconazole-sensitive C.glabrata strains of the first part of the experimental strain were broken by ultrasonic cell crusher,and total RNA was extracted by Trizol reagent.The standard strain ATCC MYA-2950 was used as the control group.The level of MSH2 gene m RNA in the experimental strain was detected by RT-q PCR.The statistical principle was used to analyze whether the level of MSH2 gene m RNA of C.glabrata was statistically significant between the fluconazole resistant group and the sensitive group.Whether there is a statistical difference in the relative amount of MSH2 gene m RNA of MSH2 gene mutation group and non-mutation group.Results The RT-q PCR amplification of each experimental strain,the relative amount of MSH2 gene m RNA in the fluconazole resistant group 2-??CT value compared with the sensitive group,the difference was statistically significant(P <0.05),and relatively low level of MSH2 gene m RNA in the drug resistance group;among the 15 MSH2 gene unmutated strains group,the percentage of MSH2 gene m RNA relatively low level strain was 33.33%(5/15),and among the 27 MSH2 gene mutated strains group,the relatively low level of strains accounted for 88.89%(24/27),the frequency of low m RNA level in the MSH2 gene unmutated group(33.33%)was significantly different from the MSH2 gene mutation group(88.89%)(P<0.05).Conclusion Fluconazole resistance is associated with low m RNA level of MSH2 gene.MSH2 gene mutations will cause low level transcription of MSH2 gene,which may be the molecular basis of MSH2 gene play a role in the resistance mechanism of C.glabrata.