[FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS]

Abstract

Objective: To explore the function of intercellular adhesion A (icaA), fibrinogen binding protein (fbe), and accumulation-associated protein (aap) genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms.

Methods: The experiment was divided into 3 groups: single culture of Staphylococcus epidermidis ATCC35984 (S. epidermidis group) or Candida albicans ATCC10231 (C. albicans group), and co-culture of two strains (mixed group) to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2, 4, 6, 8, 12, 24, 48, and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy (SEM) was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of icaA, fbe, and aap genes were analyzed by real-time fluorescent quantitative PCR.

Results: Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group, and there was significant difference between 2 groups at the other time (P < 0.05) except at 72 hours (P > 0.05). In C. albicans group, the biofilm started to grow at 12 hours of cultivation, but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points (P < 0.05). XTT assay showed that the overall growth speed in the mixed group was greater than that in the C. albicans group, and it was greater than that in the S. epidermidis group at 48 hours; there was no significant difference in the growth speed between the mixed groups and the S. epidermidis group in the other time points (P > 0.05) except at 12 hours (P < 0.05). The absorbance (A) value in the mixed group was lower than that in the S. epidermidis group at 2 and 4 hours, but no significant difference was shown (P > 0.05); the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours (P < 0.05). SEM observation showed that mature biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe, icaA, and aap genes in mixed group increased 1.93, 1.52, and 1.46 times respectively at 72 hours compared with the S. epidermidis group (P < 0.05).

Conclusion: Mixed species biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans, which is related to increased expressions of the icaA, fbe, and aap genes of Staphylococcus epidermidis.