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.
Objective To study the influence of brominated furanones on the biofilm formation of Escherichia coli on the polyvinyl chloride (PVC) material, and to provide new ideas for the research of surface modification of materials and cl inicaltreatment of biomaterial centered infection. Methods Three brominated furanones with representative chemical structurewere chosen and coated on the surface modification of PVC materials, respectively [furanone 1: 3, 4-dibromo-5-hydroxy-furanone; furanone 2: 4-bromo-5-(4-methoxyphenyl)-3-(methylamino)-furanone; furanone 3: 3, 4-dibromo-5, 5-bis (4-methylphenyl)- 2 (5H)-furanone]. All the modificated PVC materials and Escherichia coli were co-cultivated. The PVC material soaked with 75% ethanol for 5 minutes and Escherichia coli were co-cultivated together as the control group. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM). Results The CLSM showed that the thickness of bacterial community and the bacterial community quantity in the unit area of PVC materials was significantly less (P lt; 0.05) in furanone 3 group than in control group, but no significant difference (P gt; 0.05) was found between furanone 1, furanone 2 groups and control group. SEM showed that the quantity of bacterial community in the unit area of PVC materials surface in furanone 3 group was fewer than that in control group at 6 hours; the biofilm structure on PVC materials surface formed at 18 hours in control group, furanone 1 group, and furanone 2 group, but there was no mature biofilm structure on PVC materials surface in furanone 3 group at 18 hours. Conclusion The impact of different brominated furanones on Escherichia coli biofilm formation on the surface of PVC materials is different, 3, 4-dibromo-5, 5-bis (4-methylphenyl)-2 (5H)- furanone can inhibit Escherichia coli biofilm formation on the surface of PVC material.
Abstract: Objective To investigate the effect of cytokineinduced killer (CIK) cells immunotherapy on immunity function of non-small cell lung cancer (NSCLC) patients after operation. Methods Fifty patients with histological or cytological diagnosis of NSCLC on Ⅰstage,Ⅱstage andⅢa stage of tumor, nodes, metastasisclassification were randomly divided CIK cells therapy group and conventional therapy group, 25 cases each group. The immunity function of patients with NSCLC, including the levels of CD3+, CD4+ T cells, ratio of CD4+/CD8+, natural killer(NK) cells, and the levels of Th1/Th2 cytokine were detected before treatment, and the 2nd, 4th, 8th week after treatment. Results The levels of CD3+, CD4+ T cells, NK cells, ratio of CD4+/CD8+, interleukin-2(IL-2), interferon-γ(INF-γ) in CIK cells therapy group at the 2nd week after treatment were more higher than those before treatment (Plt;0.01), their levels reached the peak at 4th week, from then on, it began to decrease. Meanwhile, the levels of Th2 of CIK cells therapy group began to decrease at the 2nd week after treatment, a low ebb at 4th week. At the 2nd, 4th and 8th week,the levels of CD3+,CD4+ T cells, ratio of CD4+/CD8+, NK cells,IL-2, INF-γ, interleukin-4(IL-4), interleukin-10(IL-10) of CIK cells therapy group compared with those inconventional therapy group,there were statistical significance difference［(Plt;0.05),at 4th week after treatment, CD3+ 70.2%±9.1% vs.46.3%±5.8%; CD4+40.2%±7.1% vs.22.9%±4.5%; CD4+/CD8+ 1.82±0.43 vs. 1.09±0.34; NK 15.7%±5.4% vs.10.5%±2.5%; IL-2 34.8±11.7 ng/L vs. 19.8±12.1 ng/L; INF-γ63.7±23.3 ng/Lvs. 30.8±10.6 ng/L; IL-4 10.2±8.6 ng/L vs. 25.8±6.3 ng/L; IL-10 10.6±3.4 ng/L vs. 21.4±8.6 ng/L］. Conclusion The results indicate that CIK cells immunotherapy can enhance the immune function of NSCLC patients after operation.
ObjectiveTo explore the influence of three central venous catheter biomedical materials (polyurethane, silicone, and polyvinyl chloride) on the proliferation, apoptosis, and cell cycle of Xuanwei Lung Cancer-05 (XWLC-05) cells so as to provide the basis for clinical choice of central venous catheter. MethodsXWLC-05 cells were cultured and subcultured, and the cells at passage 3 were cultured with polyurethane, silicone, and polyvinyl chloride (1.0 cm × 1.0 cm in size), and only cells served as a control. At 24, 48, and 72 hours after cultured, MTT assay was used to detect the cellular proliferation and flow cytometry to detect the cell cycle and apoptosis. At 72 hours after cultured, inverted microscope was used to observe the cell growth. ResultsInverted microscope showed the cells grew well in control group, polyurethane group, and silicone group. In polyvinyl chloride group, the cells decreased, necrosed, and dissolved; residual adherent cells had morphologic deformity and decreased transmittance. At 24 and 48 hours, no significant difference in proliferation, apoptosis, and cell cycle was found among 4 groups (P gt; 0.05). At 72 hours, the proliferations of XWLC-05 cells in three material groups were significantly inhibited when compared with control group (P lt; 0.05), and the cells in polyvinyl chloride group had more significant proliferation inhibition than polyurethane group and silicone group (P lt; 0.05), but there was no signifcant difference in proliferation inhibition between polyurethane group and silicone group (P gt; 0.05). Compared with the control group, three material groups had significant impact on the rate of apoptosis and cell cycle: polyvinyl chloride group was the most remarkable, followed by silicone group, polyurethane group was minimum (P lt; 0.05). ConclusionPolyvinyl chloride can significantly impact the proliferation, apoptosis, and cell cycle of XWLC-05 cells; polyurethane has better biocompatibility than polyvinyl chloride and silicone
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 study the effect of intercellular adhesion (ica) operon of Staphylococcus epidermidis on the inflammation associated with mixed biofilm of Staphylococcus epidermidis and Candida albicans on endotracheal tube material in rabbits. MethodsThe standard strains of Staphylococcus epidermidis RP62A (ica operon positive, positive group) and ATCC12228 (ica operon negative, negative group) were taken to prepare a bacterial solution with a concentration of 1×106 CFU/mL, respectively. Then, the two bacterial solutions were mixed with the standard strain of Candida albicans ATCC10231 of the same concentration to prepare a mixed culture solution at a ratio of 1∶1, respectively. The mixed culture solution was incubated with endotracheal tube material for 24 hours. The formation of mixed biofilm on the surface of the material was observed by scanning electron microscope. Thirty New Zealand rabbits, aged 4-6 months, were divided into two groups (n=15), and the endotracheal tube materials of the positive group and the negative group that were incubated for 24 hours were implanted beside the trachea. The body mass of rabbits in the two groups was measured before operation and at 1, 3, and 7 days after operation. At 1, 3, and 7 days after operation, the levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and monocytechemotactic protein 1 (MCP-1) were detected by using an ELISA test kit. At 7 days after operation, the formation of mixed biofilm on the surface of the endotracheal tube materials was observed by scanning electron microscope, the inflammation and infiltration of tissues around the materials were observed by HE staining, and the bacterial infections in heart, lung, liver, and kidney were observed by plate colony counting method.ResultsScanning electron microscope observation showed that the mixed biofilm structure was obvious in the positive group after 24 hours in vitro incubation, but no mixed biofilm formation was observed in the negative group. In vivo studies showed that there was no significant difference in body mass between the two groups before operation and at 1, 3, and 7 days after operation (P>0.05). Compared with the negative group, the levels of MCP-1 and IL-1β at 1 day, and the levels of IL-1β, MCP-1, IL-6, and TNF-α at 3 and 7 days in the positive group all increased, with significant differences (P<0.05). Scanning electron microscope observation showed that a large amount of Staphylococcus epidermis and mixed biofilm structure were observed in the positive group, and a very small amount of bacteria was observed in the negative group with no mixed biofilm structure. HE staining of surrounding tissue showed inflammatory cell infiltration in both groups, and neutrophils and lymphocytes were more in the positive group than in the negative group. There was no significant difference in the number of bacterial infections in heart and liver between the two groups (P>0.05). The number of bacterial infections in lung and kidney in the positive group was higher than that in negative group (P<0.05).ConclusionIn the mixed infection of Staphylococcus epidermidis and Candida albicans, the ica operon may strengthen the structure of the biofilm and the spread of the biofilm in vivo, leading to increased inflammatory factors, and the bacteria are difficult to remove and persist.
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.