ObjectiveTo investigate biofilm formation on the surface of silica gel by breast surgery clinical specimens of Staphylococcus epidermidis and to analyze the relationship between biofilm formation and icaA, icaD, and accumulation-associated protein (aap) gene. MethodsBetween December 2011 and January 2013, 44 strains of Staphylococcus epidermidis were isolated from the clinical specimens of the female patients who had no symptom of infection. The icaA, icaD, and aap genes were detected by PCR and 4 genotypic groups were divided:icaA+icaD+/aap+ group (group A), icaA+icaD+/aap- group (group B), icaA-icaD-/aap+ group (group C), and icaA-icaD-/aap- group (group D). Biofilms mass was semi-quantified by semi-quantitative adherence assay after 8, 12, 24, 30, and 36 hours of incubation. The thickness of biofilms was measured by confocal laser scanning microscope (CLSM) at 12 and 24 hours after incubation. The ultrastructure of biofilms was observed by scanning electron microscope (SEM) at 24 hours after incubation. ResultsPCR test showed that 13 strains were icaA+icaD+/aap+(group A), 12 strains were icaA+icaD+/aap-(group B), 16 strains were icaA-icaD-/aap+(group C), and 3 strains were icaA-icaD-/aap-(group D). In 29 strains which had bacterial biofilm formation (65.9%), there were 13 strains in group A, 7 strains in group B, 9 strains in group C, and 0 in group D. The result of semi-quantitative adherence assay showed no significant difference in the absorbance (A) values among 4 groups at 8 hours (P>0.05). The A values of groups A, B, and C were significantly higher than that of group D at 12-36 hours, and group A was significantly higher than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The results of CLSM showed that the thickness of biofilm in groups A, B, and C was significantly larger than that in group D at 12 and 24 hours after incubation (P<0.05), and the thickness of biofilm in group A was significantly larger than that in groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The result of SEM showed that the mature biofilm could be observed on the surface of silica gel in groups A, B, and C, and the ultrastructure of biofilms in group A were the most abundant and extensive among 3 groups. The ultrastructure of biofilm in group B was similar to that in group C. No obvious biofilms formed in group D. ConclusionicaA, icaD, and aap genes all play key roles in the process for biofilm formation of Staphylococcus epidermidis. Futhermore, aap gene enhance the ability of biofilm-forming when aap and ica genes coexist, so the biofilm-forming ability of icaA+icaD+/aap+ is strongest.
ObjectiveTo 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. MethodsThe 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. ResultsCrystal 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). ConclusionMixed 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.