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.
ObjectiveTo establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm on polyvinyl chloride (PVC) material, and to observe mixed species biofilm formation and its microstructure. MethodsStaphylococcus epidermidis bacteria (ATCC35984) and Candida albicans fungal (ATCC10231)were co-incubated with 0.5 cm diameter PVC pieces in tryptic soy broth (TSB) to form mixed specie biofilms (experimental group). At 2, 6, 12, 24, 48, and 72 hours, the thicknesses of the biofilms, the number of bacteria per sight, and the percentage of viable cells in biofilms were measured, and three-dimensional images of biofilms were obtained using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) at 48 hours. PVC material cultured in the TSB medium served as control group. ResultsIn control group, there was no pathogenic bacteria adhesion on the PVC material surface. In experimental group, CLSM showed that colonies and biofilm formation were found at 6 hours after co-culture, and gradually increased with time. The pathogenic bacteria colonies reached the peak at 24 hours, and biofilm thickness attained peak value at 48 hours. In experimental group, the number of colony was significantly different among 2, 6, and 24 hours, and between 2, 6 hours and 48, 72 hours (P<0.05), but no significant difference was found among 24, 48, and 72 hours (P>0.05). The biofilm thickness showed significant difference between the other time points (P<0.05) except between 48 and 72 hours (P>0.05). The percentage of viable cells in the outer layers of the biofilm was significantly higher than that in inner and middle layers at 48 hours (P<0.05). Three-dimensional reconstruction displayed that the surface of mixd species was uneven; living bacterium mainly located at the protuberance, and dead bacteria mainly located at the concaves. SEM image showed that Staphylococcus epidermidis attached to various forms of Candida albicans (spores, pseudohyphae, hyphae) gradually, and formed multilayer reticulate sophisticated structure on the surface of PVC with time. ConclusionCandida albicans-Staphylococcus epidermidis mixed species biofilm is sophisticated in structure. The combination of CLSM, SEM, and three-dimensional image reconstruction technology is ideal for investigation of mixed species biofilm on PVC material.
ObjectiveTo investigate the effect of the estradiol hormones on biofilm formati on and structure of Staphylococcus epidermidis after breast implant surgery. MethodsThe concentration of Staphylococcus epidermidis strains ATCC35984 was adjusted to 1×107 CFU/mL or 1×108 CFU/mL, and the type strains were incubated on the surface of silica gel in 125 pmol/L estradiol suspensions to prepare bacterial biofilms model in vitro. After cultured in vitro for 4, 6, 12, 24, 48, and 72 hours, bacteria growth and biofilm formation ability were assessed by means of the XTT and crystal violet staining respectively. According to the above results, the bacterial suspension concentration was selected for experiments. The experimental concentration of Staphylococcus epidermidis ATCC35984 suspension and the concentrations of 50, 125, 250, 500 pmol/L estradiol suspensions were mixed with silica gel respectively to prepare biofilm model in vitro, no estradiol suspension served as control group. The experimental concentration of Staphylococcus epidermidis ATCC12228 suspension was used to prepare the same model in the negative control. After cultured in vitro for 4, 6, 12, 24, 48, and 72 hours, the same methods were used to assess the bacteria growth dynamics and biofilm forming ability, and the scanning electron microscope (SEM) was used to observe bacterial biofilm structure cultured on the surface of silica gel; the laser scanning confocal microscope (CLSM) was used to measure bacterial biofilm thickness on the surface of silica gel after 6, 12, and 24 hours. ResultsAccording to the results of semi quantitative detection of crystal violet stain and XTT methods, the bacterial suspension of 1×107 CFU/mL was selected for the experiment. XTT results indicated that the growth rates of ATCC12228 strain (at 4, 6, 12, 24, and 72 hours) and ATCC35984 strain (at 4, 6, 24, and 72 hours) in 125, 250, and 500 pmol/L estradiol were significantly faster than those in 0 and 50 pmol/L (P < 0.05). The growth rate of 500 pmol/L group was significantly faster than 125 and 250 pmol/L groups at 4, 6, and 72 hours (P < 0.05), and the growth rate of 250 pmol/L group was significantly faster than that of 125 pmol/L group at 72 hours (P < 0.05), but there was no significant difference between 0 and 50 pmol/L groups (P>0.05). At the same time point and same estradiol concentration, the growth rates showed no significant difference between 2 strains (P>0.05). Semi quantitative detection of crystal violet staining showed no biofilm formed in ATCC12228 strain in all estradiol concentration groups at different time points. In ATCC35984 strain, the biofilm was found at 4 hours and gradually thickened with time, reached the peak at 24 hours. After cultured for 4 and 6 hours, the biofilm of 0 pmol/L groups were significantly thicker than that of 125, 250, and 500 pmol/L groups (P < 0.05). At 12 hours, the 125 pmol/L group had the thickest biofilm, showing significant difference when compared with other groups (P < 0.05). The CLSM showed ATCC35984 biofilm thickness of 125, 250, and 500 pmol/L was significantly less than that of 0 and 50 pmol/L groups at 6 hours (P < 0.05), but difference was not significant between other groups (P>0.05). Then the thickness of the biofilm increased gradually, and the thickness of 125 pmol/L group was significantly larger than that of other concentration groups at 12 and 24 hours (P < 0.05). The SEM observation showed that the biofilm of 125 pmol/L group was denser and thicker than that of the other concentration groups at each time point. ConclusionHigh level estradiol can promote bacteria growth, biofilm formation, and biofilm maturity of Staphylococcus epidermidis.
Xuanwei district in Yunnan Province of China has pretty high incidence of lung cancer in China, even around the world. Studies have shown that there exists a close relationship between lung cancer and local indoor air pollution caused by Bituminous coal. Considering that the indoor air pollution in Xuanwei District is caused by "open fireplace", an indoor air pollution simulation system was designed, and an F344 rats lung damage model was established for this indoor air pollution fireplace. The model is based on indoor air pollution simulation system with signal multiplexer control and multichannel acquisition, and mining PID algorithm was used for polynomial fitting to each test point, and a relatively constant PM2.5 air pollution status was simulated. The results showed that the system could simulate a variety of states of air pollution, provide a new test method for evaluation of human injury caused by indoor air pollution and a new idea for the study of the incidence of lung cancer in Xuanwei district and other places.