Azole Resistance and Detection of the ERG11 Gene in Clinical Candida albicans Isolated from Pregnant Women with Vulvovaginitis Attending Federal Medical Centre, Yenagoa, Nigeria

Introduction:Candida albicansis one of the most important aetiological agents causing vaginal candidiasis in pregnant women.Most women will experience at least one episode during their reproductive years. Antifungal resistance is a particular problem with Candida infections. Some types of Candida are increasingly resistant to the first-line and second-line antifungal medications.Objective:To investigate the azole susceptibility of Candida albicans(C. albicans) from pregnant vulvovaginal candidiasis patients and to detect ERG11gene in these azole resistance isolates.Methods:Forty-one clinical isolates of C. albicanswere collected. Azole susceptibility was tested in vitrousing microdilution techniques.The ERG11genes of 27 isolates of C. albicans(All resistant to azoles) were amplified using PCR method

Two natural molecules preferentially inhibit azole-resistant Candida albicans with MDR1 hyperactivation / 中国天然药物

Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B (RAB) and riccardin D (RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.

Azole Resistance Caused by Increased Drug Efflux in Candida glabrata Isolated from the Urinary Tract of a Dog with Diabetes Mellitus

A yeast-like organism was isolated from a urine sample of a 6-year-old neutered male miniature poodle dog with urinary tract infection, diabetes ketoacidosis, and acute pancreatitis. We identified the yeast-like organism to be Candida glabrata and found that this fungus was highly resistant to azole antifungal drugs. To understand the mechanism of azole resistance in this isolate, the sequences and expression levels of the genes involved in drug resistance were analyzed. The results of our analysis showed that increased drug efflux, mediated by overexpression of ATP transporter genes CDR1 and PDH1, is the main cause of azole resistance of the C. glabrata isolated here.

Mechanism of azole resistance in the Candida albicans strains isolated from vulvovaginal candidiasis / 中国感染与化疗杂志

Objective To investigate the resistance rates of the Candida albicans strains isolated from patients with vulvovaginal candidiasis to 5 antifungal agents and examine the mechanism of azole resistance in these strains.Methods A total of 1 646 C.albicans strains were collected in Obstetrics and Gynecology Hospital of Fudan University from January to December 2015.The resistance rates of these isolates to five antifungal agents were analyzed.Azole-resistant (n=30),dose dependent sensitive (S-DD) (n=13),and susceptible isolates (n=10) were randomly selected from the microbiology laboratories of three obstetrics and gynecology hospitals in Shanghai.The expression levels of drug efflux pump related gene CDR1,CDR2,MDR1 and drug target enzyme gene ERG11 were analyzed by real-time fluorescence quantitative polymerase chain reaction (PCR).At the same time,the ERG11 and ERG3 genes were amplified by PCR and sequenced,and analyzed for resistance-related mutations.Results Of the 1 646 C.albicans strains,5.2％,3.2％,2.5％ and 2.1％ were resistant to itraconazole,voriconazole,fluconazole and 5-fluorocytosine,respectively.All isolates were sensitive to amphotericin B.The expression of ERG11 gene was significantly higher in S-DD group and azole-resistant group than in azole-sensitive group (P＜0.05).The expression of CDR1,CDR2 and MDR1 did not show significant difference among the three groups.There were 13 missense mutations in the ERG11 gene,of which T123I,P98S and Y286D amino acid substitutions were newly discovered.Both T123I and Y132H were identified in 26 resistant isolates,of which 16 gene mutation was detected in two pan-azole-resistant isolates.Conclusions The C.albicans strains isolated from vulvovaginal candidiasis showed higher resistance rates to azole antifumgal agents than that to 5-fluorocytosine and amphotericin B.Mutation and over-expression ofERG11 gene may be one of the prevalent molecular mechanisms underlying azole resistance in C.albicans.were pan-azole-resistant.In addition,the ERG3 heterozygous

Synergistic effects of tacrolimus and azole antifungal compounds in fluconazole-susceptible and fluconazole-resistant Candida glabrata isolates

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata, a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro, notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

Mutation of P927S in PRD1 gene mediates azole resistance in Candida glabrata / 中华传染病杂志

Objective To investigate the role of PDR1 gene in azole-resistant Candida glabrata (C.glabrata).Methods Thirty-eight clinical isolates of C.glabrata were collected from five different hospitals.The minimal inhibitory concentrations (MIC) of azole antifungals including fluconazole,itraconazole and voriconazole against C.glabrata were determined by broth microdilution.Sequencing and amplification of PDR1 gene was achieved by real-time quantitative polymerase chain reaction (PCR).The mutation was cloned into an expression plasmid and then transferred into C.glabrata.The efflux of rhodamine 6G and drug sensitivity test were performed,and expressions of CDR1 and CDR2 were examined to verify function of mutation.Results Among these 38 isolates of C.glabrata,17 were resistant to at least one of azole antifungals.Moreover,mutations of PDR1 gene existed in every resistant isolates.Results of phenotyping test showed that in the isolate that expressed PDR1P927S,the expression of CDR1 and CDR2 were increased by 20.53 and 4.03 fold,respectively.And the fluorescence intensity of rhodamine 6G was decreased to 0.62 in efflux experiment.Conclusion P927S mutation of PDR1 gene could induce azole resistance of C.glabrata by increasing the expressions of CDR1 and CDR2,which results in drug resistance due to enhanced effect of efflux pump.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis / 대한진단검사의학회지

BACKGROUND: Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species. METHODS: The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C. haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates. RESULTS: The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3+/-18.5, 126.9+/-6.6, 88.5+/-18.5, 50.8+/-7.0, and 38.1+/-3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF. CONCLUSIONS: This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.

Membrane fluidity and lipid composition of fluconazole resistant and susceptible strains of Candida albicans isolated from diabetic patients / Fluidez e composição lipídica da membrana de cepas de Candida albicans resistentes e sensíveis ao fluconazol, isoladas de pacientes diabéticos

Ten clinical isolates of Candida albicans, five strains belonging to each of fluconazole resistant and susceptible groups isolated from diabetic patients, were studied for the membrane fluidity and lipid composition. Compared to fluconazole susceptible strains, fluconazole resistant ones exhibited enhanced membrane fluidity as measured by fluorescence polarization technique. The increased membrane fluidity was reflected in the decreased p-values exhibited by the resistant strains. On the other hand, susceptible isolates contained higher amount of ergosterol, almost twice as compared to resistant isolates which might have contributed to their lower membrane fluidity. However, no significant alteration was observed in the phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of the exposed aminophospholipid, phosphatidylethanolamine was highest in the resistant strains as compared to the susceptible strains, indicating a possible overexpression of CDR1 and CDR2 genes in resistant strains. The results presented here suggest that the changes in the ergosterol content and overexpression of ABC transporter genes CDR1 and CDR2 could contributeto fluconazole resistance in C. albicans isolated from diabetic patients.

Dez isolados clínicos, sendo cinco resistentes e cinco sensíveis ao fluconazol, obtidos de pacientes diabéticos, foram estudados quanto à fluidez e composição química da membrana. Quando comparados aos isolados sensíveis ao fluconazol, os isolados resistentes apresentaram fluidez de membrana aumentada, conforme mensurado pela técnica de polarização fluorescente. A fluidez de membrana aumentada refletiu-se pelos valores mais baixos de p. Por outro lado, os isolados sensíveis continham quantidades mais elevadas de ergosterol, quase o dobro dos isolados resistentes, o que pode ter contribuído para a fluidez de membrana mais baixa. Entretanto, não se observou alteração significativa na composição fosfolipídica e de ácidos graxos nesses isolados. Experimentos de marcação com corante fluorescamina indicaram que a porcentagem de aminofosfolípides e fosfatidiletanolamina expostos foi mais elevada nos isolados resistentes do que nos sensíveis, indicando uma possível superexpressão dos genes CDR1 e CDR2 nos isolados resistentes. Os resultados aqui apresentados sugerem que alterações no teor de ergosterol e superexpressão dos genes ABC transportadores CDR1 e CDR2 podem contribuir na resistência ao fluconazol em isolados de C. albicans de pacientes diabéticos.

Relationship Between Gene CYP51 and Clinical Azole-resistant Candida albicans Isolates / 中华医院感染学杂志

OBJECTIVE To explore the relationship between the point mutations of gene CYP51 and the azole-resistance mechanism in clinical Candida albicans isolates.METHODS The paper diffusion test and NCCLS M-27 protocols were used to screen the fluconazole-resistant and itraconazole-resistant C.albicans clinical isolates.Gene CYP51 of two azole-resistant C.albicans clinical strains(2007H and 2007T strains) was amplified by three pairs of primers,respectively.The PCR products purified were sequenced,and compared with the nucleotide sequences of C.albicans(accession No.:X13296) to find out the mutation sites.RESULTS The nucleotide sequence analysis showed that there were both significant point mutations and silent mutations in gene CYP51 from two azole-resistant isolates of C.albicans.Seven mutations previously described,F105L,K128T,Y132H,T199I,R267H,G464S,and R467K,were identified in the two strains.The animo acid substitutions of Y132H and R467K,known to contribution to azole resistance,were detected in both 2007H strain and 2007T strain.Four novel mutations,including F71L,W244R,T311N and T352I,were simultanously identified.Nine silent mutations appeared in two isolates.CONCLUSIONS In this survey,the two azole-resistant C.albicans clinical isolates contained more than one mutation in gene CYP51 that is associated with azole resistance.Four novel mutations of CYP51 may be associated with the resistance of C.albicans to azoles.And the mechanisms need to be further studied in detail.