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.
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.
Objective To overview the effect of bacterial biofilms (BBF) on the formation of chronic osteomyel itis and the treatment measure. Methods The original articles in recent years about the relationship between BBF and chronic osteomyel itis were reviewed. Results The diagnosis and treatment of chronic osteomyel itis was very difficult, besides hyperplasia oflocal scar, poor blood supply, drug-resistant, forming of BBF also was an important reason. BBF formed on the surface of necrosis soft tissue and dead bone. Due to the protection of BBF, the bacterium were far more resistant to antimicrobial agents, which caused the recurrence of chronic osteomyel itis. The forming of BBF included three processes which were adhesion, development and maturity. As the major pathogens of chronic osteomyel itis, staphylococcus had its own characteristic. Designing therapeutic programmes according to these characteristics had become the trend of anti-infection treatment of BBF. Conclusion Although there are lots of studies on anti-biofilm due to the key factors during the forming of BBF, the most effective way of anti-biofilm is still debridement.
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.
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.
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 investigate the effect of accessory gene regulator C (agr C) specific binding peptides (named N1) on the biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride (PVC) materials in vitro.MethodsFirstly, the two strains (ATCC35984, ATCC12228) were cultured with N1 at concentrations of 100, 200, 400, 800, and 1 600 μg/mL, respectively. The control group was cultured with agrC specific binding unrelated peptides (named N0) at the same concentrations and the absorbance (A) value was measured after 24 hours to determine the optimal bacteriostatic concentration of N1. The two strains were cultured with N1 and N0 of the optimal concentration, respectively. The A values were measured at 6, 12, 18, 24, 30, and 48 hours to observe the effect of N1 on the biofilm formation ability of Staphylococcus epidermidis. On this basis, the surface structure of the biofilm on the surface of PVC material was observed by scanning electron microscopy after 6, 12, 18, 24, and 30 hours of incubation with PVC material sheet. The thickness of the biofilm was observed by laser confocal microscopy after 6, 12, 18, and 24 hours of incubation with ATCC35984 strain.ResultsThe optimal bacteriostatic concentration of N1 was 800 μg/mL. ATCC 12228 strain did not form obvious biofilm after being cultured with N1 and N0. When ATCC35984 strain was cultured with N1 and N0 for 12 hours, the difference in biofilm formation ability between groups N1 and N0 was statistically significant (P<0.05), but there was no significant difference at 6, 18, 24, 30, and 48 hours (P>0.05). Scanning electron microscopy examination showed that mature biofilm structure was observed in ATCC35984 strain and was not observed in ATCC12228 strain. Laser confocal microscopy observation showed that the number of bacteria in the group N1 was significantly lower than that in the group N0 at 12 hours, and the most of bacteria were dead bacteria. There was no significant difference in the number of bacteria at 6, 18, and 24 hours, and the most of them were live bacteria. The biofilm thickness of group N1 was significantly lower than that of group N0 at 12 and 18 hours (P<0.05).ConclusionThe intensity of N1 inhibiting the formation of Staphylococcus epidermidis biofilm is dose-dependent. During the aggregation period, N1 can inhibit the biofilm formation by hindering the bacterial growth and aggregation. The inhibition effect on mature biofilm is not obvious.
Objective It is difficult to treat chronic osteomyel itis due to the formation of the Staphylococcus aureus biofilms. Liposomal gentamicin-impregnated allogeneic cortical bone can inhibit the formation of the Staphylococcus aureusbiofilms. To explore the treatment of chronic osteomyel itis of rabbit by l iposomal gentamicin-impregnated allogeneic cortical bone. Methods The l iposomal gentamicin, l iposomal gentamicin-impregnated allogeneic cortical bone and gentamicinimpregnated allogeneic cortical bone were produced. Then the chronic Staphylococcus aureus osteomyel itis models of rabbit were made in left lower l imbs of 40 6-month-old rabbits and the right lower l imbs were used as controls. After 2 weeks, the observations of gross and X-ray were done. Four rabbits died within 10 days after the models were made and other 36 rabbits were devided into 6 groups: group A (no antibiotics), group B (intravenous injection of gentamicin), group C (intravenous injection of l i posomal gentamicin), group D (implantation of gentamicin-impregnated allogeneic cortical bone), group E (implantation of l i posomal gentamicin-impregnated allogeneic cortical bone), and group F (implantation of allogeneic cortical bone). After 2 weeks of treatment, the bacterial culture, X-ray and HE staining were done. Results The chronic Staphylococcus aureus osteomyel itis model of rabbit was made successfully. The X-ray showed dissolution of bone and periosteal reaction in groups A, B, C, and F, and no obvious dissolution of bone and periosteal reaction in groups D and E. The Norden scores were (2.5 ± 0.3), (2.1 ± 0.2), (1.5 ± 0.3), (1.5 ± 0.2), (0.9 ± 0.3), and (2.7 ± 0.3) points in groups A-F, respectively; showing significant differences between group A and groups B-E (P lt; 0.05), between groups B, E, F and other groups (P lt; 0.05). The results of blood and marrow cultures for Staphylococcus aureus were positive in groups A and F, and negative in other 4 groups; the results of bone marrow culture for Staphylococcus aureus were positive in 6 rabbits of group B, 4 rabbits of group C and 3 rabitts of group D; and the results were negative in group E. HE staining showed: in groups A and F, abscess and dead bone formed, and no new bone formation were observed; in groups B and C, different degrees of neutrophil accumulation was seen; in group D, some neutrophil accumulation occurred, and osteoprogenitor cells and osteoclasts were seen around implanted bone; and in group E, no neutrophil accumulation was observed, a lot of granulation tissues formed, and osteoprogenitor cells and osteoclasts were seen around implanted bone. Conclusion Implantation of l iposomal gentamicin-impregnated allogeneic cortical bone has remarkly better effect in treating chronic osteomyel itis than intravenous injection of l iposomal gentamicin and implantation of gentamicin-impregnated allogeneic cortical bone.
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.