Bacterial biofilms are associated with at least 80% of human bacterial infections. The clinical treatment of biofilm infection is still arduous, and therefore many new treatment options are under study, such as probiotics and their derivatives, quorum sensing inhibitors, antimicrobial peptides, phage therapy, organic acids, light therapy, and plant extracts. However, most of these schemes are not mature, and it is important to develop new research directions of anti-biofilms.
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.
Objective The intercellular adhesion (ica) gene of Staphylococcus epidermidis (SE) is a key factor to bacterial aggregation, to analysis the genotype of iatrogenic SE and to explore the effect of iatrogenic SE ica operon on theformation of bacterial biofilm on the surface of polyvinyl chloride (PVC). Methods Fifty-six cl inical isolates of iatrogenic SEwere selected, and PCR and gene sequencing were used to detect the genes related with bacterial biofilm formation. The genes contained 16S rRNA, autolysin (atlE), fibrinogen binding protein (fbe), and icaADB. The bacteria suspension of 1 × 105 cfu/mL iatrogenic SE was prepared; according to the test results of target genes, the PVC material and the genotype of icaADB+, atlE+, fbe+ strains were co-cultivated as the ica positive group; the PVC material and the genotype of icaADB-, atlE+, fbe+ strains were co-cultivated as the ica negative group. The thickness of biofilm and bacterial community quantity unit area on PVC materials were measured by confocal laser scanning microscope, and the surface structure of biofilm formation was observed by scanning electron microscope (SEM) at 6, 12, 18, 24, and 30 hours. Results The positive rate of 16S rRNA of iatrogenic SE strains was 100% (56/56). The genotype of icaADB+, atlE+, and fbe+ strains accounted for 57.1% (32/56). The genotype of icaADB-, atlE+, and fbe+ strains accounted for 37.5% (21/56). The sequencing results showed that the product sequences of 16S rRNA, atlE, fbe, and icaADB were consistent with those in GenBank. With time, no significant bacterial biofilm formed on the surface of PVC in ica operon negative group. But in ica operon positive group, the number of bacterial community was gradually increased, and the volume of bacterial biofilms was gradually increased on the surface of PVC. At 24 hours, mature bacterial biofilm structure formed, and at 30 hours, the volume of bacterial biofilms was tending towards stabil ity. The thickness of biofilm (F=6 714.395, P=0.000) and the bacterial community quantity unit area on PVC materials (F=435.985, P=0.000) in ica operon positive groupwere significantly higher than those in ica operon negative group. Conclusion Iatrogenic SE can be divided into 2 types ofica operon negative and ica operon positive bacteria. The iatrogenic SE ica operon can strengthen bacterium biofilm formation capabil ity on PVC materials, bacterium community quantity, and thickness of biofilm, it plays an important role in bacterium biofilm formation on PVC materials.
Objective To observe the inhibitory characteristics of silver nanoparticles (AgNP) on bacterial biofilms and investigate their inhibitory effect on biofilm formation on three common orthopedic biomaterials. Methods The minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC) of AgNP were determined by microplate dilution assay. Biofilms of Staphylococcus aureus (ATCC 25923) were cultured on three orthopedic biomaterials (titanium alloy, titanium oxide, and stainless steel) and intervened with AgNP at concentrations of 32, 16, 8, 4, 2 and 0 μg/mL to determine the MBICs on the three materials. The effects of AgNP on biofilm formation were analyzed by scanning electron microscopy and measuring optical density. Results The MIC and MBIC of AgNP in the microplate assay were both 16 µg/mL. The MBICs of AgNP on biofilm formation in titanium oxide, titanium alloy, and stainless steel were 16 μg/mL, 32 μg/mL, and 32 μg/mL, respectively. Among the three materials, the lowest optical density was observed on titanium oxide, while the highest was on titanium alloy. Conclusions AgNP has strong antibacterial biofilm characteristics and can prevent the formation of Staphylococcus aureus biofilm in vitro. Biofilm formation is most pronounced on titanium alloy, least on titanium oxide, and intermediate on stainless steel.
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.
ObjectiveTo evaluate the effectiveness of liquid wound dressing in the treatment of chronic ulcer wounds. MethodsBetween January 2014 and October 2015, 84 patients with chronic ulcer wounds were included and divided into 2 groups randomly. The chronic ulcer wounds were covered with liquid wound dressing in the treatment group (n=44) and were managed with iodophor in the control group (n=40). There was no significant difference in age, gender, causes, location, wound area, and disease duration between 2 groups (P > 0.05). The frequency of dress changing, effective rate of treatment, wound healing time, wound healing rate at 5, 10, and 20 days, positive rate of bacteria culture at 1, 5, and 10 days, and the rate of side effect were recorded and compared between 2 groups. Vancouver scar scale was used to evaluate scar formation. ResultsThe effective rate of the treatment group (100%) was significantly higher than that of the control group (85%) (P=0.009). The frequency of dress changing in the treatment group[(11.36±3.40) times] was significantly lower than that in the control group[(16.94±4.51) times] (t=-6.231, P=0.000). The wound healing rates at 5, 10, and 20 days were significantly increased (P < 0.05) and the wound healing time was significantly decreased (t=-6.627, P=0.000) in the treatment group when compared with the control group. The positive rates of bacteria culture at 5 and 10 days in the treatment group were significantly lower than those in the control group (χ2=12.313, P=0.000; P=0.005), but no significant difference was found at 1 day (χ2=0.066, P=0.797). Side effect was observed in 4 cases of the control group. Vancouver scar scale score was 8.59±1.32 in the treatment group and was 9.85±1.65 in the control group, showing significant difference (t=-3.752, P=0.000). ConclusionThe application of the liquid wound dressing in the treatment of chronic ulcer wound can improve the wound healing rate, shorten the healing time and decrease the frequency of dress change, which could promote the wound healing process.
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.
Objective To investigate the effect of aureolysin （Aur） on staphylococcus aureus biofilm formation of dacron biomaterial surfaces under different Aur concentration. Methods Ninety dacron biomaterials were divided into 3 groups （group A, group IA, control group） with random number table （30 piece in each group）. Dacron biomaterials were put into vials contained staphylococcus aureus （105 CFU/ml） respectively; then Aur was added to make the concentration at 400ng/ml in group A, and group B at 80ng/ml. The thickness and number of staphylococcus aureus biofilm on the surfaces of dacron biomaterials of each group were evaluated by confocal laser microscopy and scanning electron microscopy after incubating 6h, 16h, 24h, 30h, and 48h. Results The thickness and number of staphylococcus aureus biofilm on dacron biomaterials surfaces increased significantly with time dependence in control group. The thickness and number of staphylococcus aureus biofilm in group A were less than those in group B and control group at each time points （P〈0. 05）. The thickness and number in group B were significantly decreased than those in control group （P 〈 0. 05）. Conclusion The study shows that Aur can effectively inhibit the formation of staphylococcus aureus biofilm on dacron biomaterials surfaces with dose dependence.
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 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.