| Objective:Candida albicans(CA)and Staphylococcus aureus(SA)were common nosocomial pathogens and often found in polymicrobial biofilm on biotic or abiotic surfaces.The mixed-biofilm of CA & SA was related to various diseases and brought great challenges to clinical treatment.In this study,clinical isolated CA & SA were collected to reveal the characteristics of mixed-biofilm.By analyzing the influence of SA on biofilm formation and key genes expression about CA,to explore the effects and molecular regulation mechanism of CA biofilm formation,it provided new ideas for prevention and treatment of clinical mixed-biofilm infections and the development of new antibacterial drugs.Methods1.Crystal violet assay was applied to measure the biomass of single species biofilm from clinical isolated CA and SA,and the standard strain SC5314 & ATCC25923.Then,selected the CA with moderate and low biofilm formation and their co-isolated SA pairs,and the CA standard strain SC5314 with high formation and SA standard strain ATCC25923 for subsequent experiments.2.Mixed-species biofilms formed by CA with different formation ability and their co-isolated SA pairs,the biomass of mixed-species biofilms and growth curve measured by crystal violet assay,CFU level of single and mixed-species biofilms were assessed by microbiological plating on selective media.All above were utilized to explore the growth characteristics of mixed-species biofilms.3.Crystal violet assay was used to evaluate the effect of the first colonizer on mixed biofilm formation and XTT reduction assay applied to determine the change of SA supernatant on CA biofilm formation at different stage.4.Adopted fluorescence quantitative reverse transcription PCR(RT-q PCR)method to understand the expression of CA hyphae specific genes(ALS3,HWP1)and transcription factors(EFG1,BCR1,ZAP1),under the presence of SA supernatants,to understand the regulation molecular mechanism of SA on CA biofilm formation.Results:1.In this experiment,it collected 24 pairs clinical isolated CA and SA.The clinical isolated CA with high,moderate and low biofilm formation were 4.17%(1/24),45.83%(11/24),and 50.00%(12/24)respectively.As for clinical isolated SA,moderate and weak instances were 16.67%(4/24)and 83.33%(20/24).2.The biomass of biofilm showed a gradual increase over time,reached peak values at 48 h.Compared with the single-species biofilm,there was an larger quantity on the mixed-biofilm of CA & SA,with statistical significance(P < 0.05),and it contained a higher population of SA.3.Compared with the mixed-biofilm inoculated simultaneously,the biomass of mixed-species biofilm reduced significantly(P< 0.05)when SA as first colonizer and after adding CA,while no significant change when CA as first colonizer.The SA free-stated supernatant promoted the metabolic activity of CA biofilm obviously(P< 0.05),while SA biofilm supernatant promoted the growth of early biofilm of CA in various degrees and showed no significant difference in mature period.4.Compared with the CA single-species biofilm,the expression level of CA was change in various degrees when co-cultivation with SA supernatant.The expression of CA hyphae specific gene ALS3,HWP1 increased by about 2.4-fold and 1.2-fold respectively,and the biofilm transcription factor EFG was up-regulated by about 1.4-fold,while the expression of BCR1 reduced 30%,and no significant change for ZAP1.Conclusion1.The clinical isolated CA had high,moderate and low biofilm formation,while SA with low biofilm formation only.Compared with single-species biofilm,CA and SA formed a thicker,complex mixed-biofilm.2.CA and SA competed for nutrients and adhesion sites during the early period of mixed-biofilm formation,and the SA supernatant promoted the metabolic activity of CA biofilms.3.Certain SA excretion may regulate the transcription signal pathways and hyphae specific genes expression which promoted the biofilm formation of CA. |
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