A Study On Mechanisms Of Antifungal Resisitance-relate Drug Efflux Pump Genes Of Pathogenic Candida Isolates From RVVC

Objective (s):① To find out the variety of popular local distribution of candida species by isolating and identifying pathogenic strains from Recurrent Vulvovaginal candidiasis patients.② To determinate the susceptibilities of pathogenic candida species from RVVC, non-candida albicans additionally, to seven antifungal drugs in vitro.③ To identify expression of the ATP binding cassette transporters, CDR1, CDR2,PDR1 and the multi-drug resistance MDR1, and to confirm the role of efflux pumps genes in antifungal resisitance of C.albicans and non-candiada albicans.Methods:① All 190 pathogenic isolates of Candidiasis collected from our hospital were isolated and identified by the VITEK2 system(bioMerieux), including 99 isolates from RVVC and 91 isolates from VVC; ② All identified 190 isolates were tested in vitro to seven antifungul drugs including two kinds of Polyene antifungul drugs,four kinds of azoles and 5-fluoroctosine by BIOKONT fungus kits (FUNGUS-7); ③ All isolates divided into susceptible, susceptible dose dependent and resistant groups were amplified with realtime fluores-cence quantitative PCR (RTFQ PCR) to study the expression difference of the four drug efflux pump related genes,CDR1,CDR2,PDR1,MDR1, from different groups.Results: ① The proportion of candida albicans is 72.10%(137/190) and the proportion of non-candida albicans is 27.89%(53/190) in all candida isolates.Candida glabrata and Candida tropicalis are the most common non-candida albicans, the proportion are 12.10%(23/190) 11.57%(22/190) separately. There are 99 isolates in RVVC including 82 candida albicans isolates which the proportion is 82.82%, and 17 non-candida albicans isolates which the proportion is 17.18%. There is 91 isolates in VVC including 55 candida albicans which the proportion is 60.43%and 36 non-candida albicans which the proportion is 39.56%.The statistic difference between the proportion of non-candida albicans in RVVC and VVC is significant (P<0.05); ②The antifungal susceptibility order of VVC is presented as nystatin> miconazole>amphotericinB>fluconazole>itraconazole>5-fluorocytosine>econazole. RWC have the same antifungal susceptibility order,The statistic difference between the antifungal susceptibility order of RVVC and VVC has no significant difference (P>0.05). The antifungal susceptibility order of candida albicans of VVC is presented as miconazole>amphotericinB> nystatin> fluconazole> itraconazole> 5- fluorocytos-ine >econazole. The antifungal susceptibility order of non-candida albicans of VVC is presented as nystatin> amphotericinB>miconazole,5-fluorocytosine> fluconazole> itraconazole>econazole. The candida albicans show a higher sensibility to miconazole, fluconazole, itraconazole than non-candida albicans in RVVC(P<0.05). ③Comparing the expression difference of four drug efflux pump related gene PDR1, CDR1, CDR2, MDR1, among group WC and group RVVC, the results are shown that 36 candida isolates express PDR1, including 24 candida albicans and 12 non-candida albicans which include 7 candida glabrata strains having a proportion of 30.43%(7/23),2 candida tropicalis strains having a proportion of 9.09%(2/22),2 candida parapsilosis strains having a proportion of 50%(2/2). In WC,there are 14 candida albicans and 10 non-candida albicans expressing PDR1 which the proportion s are 25.45%(14/55) and 27.78%(10/36) separately. In RVVC,there are 10 candida albicans and 2 non-candida albicans expressing PDR1 which the proportion s are 12.20%(10/82)and 11.76%(2/17) separately. Group VVC has a higher PDR1 expression than RWC(P<0.05).Among WC’s and RWC’s R+I groups and S groups of the seven antifungal drugs, no significant differences are found in the expression strain ratio of PDR1 (P>0.05), and the comparison between VVC’s and RWC’s R+I groups and S groups shows that excepting PDRlhave a more higher expression strain ratio ineconazole VVC-R+I groups than in RWC-R+I groups,all other six drugs’VVC-S groups show a more expression of PDR1 than RVVC-S groups; To make a comparison of expression quantity of PDR1 between WC and RVVC,we foud that there are significant differences between fluconazole and econazole VVC-R,VVC-I and VVC-S groups(P<0.05), in which PDR1 have a higher expression quantity ineconazole WC-R group than in WC-I and WC-S groups(P<0.05), fluconazole also shows a higher PDR1 expression quantity in VVC-R group than in VVC-S group. Nystatin WC-S group expresses more PDR1 than RVVC-S group and Itraconazole WC-R goup expresses more PDR1 than RVVC-R group too; To compare the expression quantity of CDR1,CDR2 and MDR1 in VVC and RVVC, the results shows that RVVC-R group expresses more CDR1 quantity than RWC-I group (P<0.05). Further comparison the expression quantity between VVC and RWC showing no significant differences in CDR1,CDR2 and MDR1(P>0.05); To make a comparison in candida albicans group and non-candida albicans group of VVC and RVVC about the expression quantity of CDR1,CDR2 and MDR1 represents that candida albicans shows a higher a CDR2 expression quantity in amphotericinB I group than in amphotericinB S group in VVC (P<0.05). and non-candida albicans a higher a CDR1 expression quantity in nystin s group than in nystin R+I group in WC (P<0.05). The comparison of candida albicans and non-candida albicans in VVC about PDR1 expression quantity shows that candida albicans of VVC have a higher PDR1 expression in quantity amphotericinB, fluconazole, itraconazole,5-fluorocytosine R+I group than RVVC (P<0.05). Candida albicans have a higher CDR1 expression in group R+I of amphotericinB,nystin than non-candida albicana in WC (P<0.05) and Candida albicans have a higher MDR1 expression in group R+I of amphotericinB than non-candida albicana in VVC (P<0.05).In RVVC, CDR1 shows a more gene expression in econazole R+I group than S group (P<0.05). Candida albicans presents a more gene expression in miconazole R+I goup than non-candida albicans (P<0.05).Conclusion(s): ① Candida albicans are the main pathogenic candida spp in RWC; ② Candida glabrata and Candiada tropicals are the most common non-candida species; ③Pathogenic candida spp of VVC and RVVC have the same susceptibility order. Nystatin, miconazole,amphotericin B can be used to treat VVC and RVVC. Nystatin can be the first choice for WC and RVVC caused by non-candida albicans, while much more concentration should be payed when comes to zoles;④ CDR1,CDR2,PDR1 are possibly drug -resistance related genes of candida isolates, the over expression of CDR2 may relate to candida albicans drug resistance in VVC. the over expression of CDR1 and PDR1 may contribute to drug resistance of non-candida albicans especially the for candida glabrata when comes to fluconazole.