Objective To validate the advantage of repairing bone defect by staphylococcus aureus injection carried in collagen membrane. Methods Twentyfour adult New Zealand rabbits were divided into two groups randomly. After the experimental model of standard bone defect had been made by operation, collagen membrane/staphylococcus aureus injection and staphylococcus aureus injection with the same quantity were transplanted in bone defect areas of the two groups respectively. The reconstructed tissues were observed by general method, X-ray, histology, and immunohistochemistry at 2nd、4th、6th、8th week respectively. Results The experimental group showed that new bone proliferated distinctly in bone defect areaand the proliferation lasted long, and no excessive connective tissue in defectarea. X-ray observation showed that there was continual callus growth in transplantation area in early stage and the distribution of new bones was even in the group. Histological observation showed that there were many new bone growth centers in bone defect area, trabecular bones were sequentially distributed, and mature bone replacement was complete. Immunohistochemical examination showed that bone morphogenetic protein (BMP) could be seen for a long time and BMP took up a large part in the new bone tissues. Conclusion Collagen membrane could prevent parenchyma from penetrating into bone defect area and provide room for new bone growth. As the carrier of staphylococcus, collagen membrane could reduce the overflow of staphylococcus and improve its curative effect as well.
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.
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 construct a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A (FnBPA) r10-11 truncated fusion protein, and explore the immunogenicity of FnBPAr10-11. MethodsPloymerase chain reaction (PCR) amplification was carried out from the whole genome sequence of Staphylococcus aureus Newman strain by recombinant PCR technique. The amplified product was purified and transformed into Escherichia coli DH5α for cloning. The recombinant plasmid was extracted and identified by double enzyme digestion. The recovered fragment was ligated into the pET-32a plasmid and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The FnBPAr10-11 was purified by HIS protein purification column, identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and used to immunize mice, and the mice were divided into phosphate buffered saline (PBS) group, FnBPA group, and FnBPAr10-11 group. The serum levels of immunoglobulin G (IgG) and cytokines, and the immune protection rate of the mice were detected. ResultsSDS-PAGE result showed that the relative molecular mass of the protein was about 33.1×103. The titers of IgG antibody in FnBPAr10-11 group and FnBPA group reached 1∶128 000, and were significantly different compared with PBS group (P<0.05). The cytokine level in FnBPAr10-11 group was not significantly different compared with that in FnBPA group, and they were extremely significant (P<0.01) compared with that in PBS group. The immuno-protective effect of the FnBPAr10-11 group was over 50%. ConclusionsThe prokaryotic expression strain of Staphylococcus aureu FnBPAr10-11 truncated fusion protein was successfully constructed. The truncated protein has good immunogenicity.
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 establish rabbit models of mixture-infectious endophthalmitis induced by exogenous Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Methods A total of 84 eyes of 42 New Zealand white albino rabbits were randomly divided into 4 groups. There were 21 eyes in each group. Rabbit eyes in group 1, 2, 3 and 4 received an intravitreal injection of 0.1 ml of mix bacterium (2times;104 CFU/ ml, including 103 S. aureus and 103 E. coli), S. aureus (104 CFU/ ml), E. coli (104 CFU/ml), and sterilized saline respectively. The eyes were examined by slit-lamp microscopy, ophthalmoscopy, A/B scan, electroretinography (ERG) and bacterial culture of vitreous humors at the timepoints of 6, 12, 24, 48 and 72 hours, and 4, 7, 10, 14 days after intravitreal injection. All eyeballs were then enucleated for histopathological examination. Results Various degrees of inflammatory reactions were presented in the 3 experimental groups after the injection, and the development trend of the disease was nearly the same. In group 1 active intraocular inflammation like anterior chamber exudates, started at 12 hours after injection (which was early than that in group 2 and 3), aggravated between 48 and 72 hours, alleviated slowly from 4 to 7 days, and was obviously better after 10 to 14 days while the corneal neovascularization and vitreous gray opacity begun to form. The bacterial culture was positive in group 1 (100％, 6 hours to 14 days after injection), group 2 (100%, 6 hours to 3 days after injection) and group 3 (100% from 6 hours to 7 days, and 67.67% at 14 days after injection). It was negative for group 2 (7 to 14 days after injection) and group 4 (6 hours to 14 days after injection). The amplitude of ERG b wave dissapeard in group 1 to 3, and decreased less than 30% in group 4 from the 48th hour after injection. Histopathological examination revealed that all intraocular structures infiltrated with inflammatory cells. Conclusion Complicated endophthalmitis rabbit models can be successfully established by intravitreal injection with S. aureus and E. coli.
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.
Objective To evaluate the toxic effects of staphylococcus aureus exotoxins and neutrophils on retinal pigment epithelium (RPE) cells (RPEC). Methods An in-vitro model of bacteroidal endophthalmitis was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils in the present of staphylococcus aureus exotoxins ATCC29213. The level of lactate dehydrogenase hydroxide（LDH）in the cuture supernant was measured, and the viability of RPE was evlauated by flow cytometry and Hoechst 33342/Propidium Iodide（PI）staining. Results When RPE cells were cultured with the exotoxin ATCC29213, the LDH level and necrotic RPE cells were positive proportional to the dosage of exotoxin, but only 250mu;l or 500mu;l of ATCC29213 had a statistical significant effect. When RPE cells were co-cultured with neutrophils in the present of ATCC29213 for 6 hours, 100mu;l of ATCC29213 already had a statistical significant effect on LDH level and necrotic RPEC, and the effect was proportional to the amount of neutrophils in the culture. Conclusion Both staphylococcus aureus exotoxins and neutrophils can damage the RPEC by inducing necrosis, and their function had synergetic effect.
ObjectiveTo analyze the characteristics of distribution and drug resistance of clinical isolated staphylococci in the Whire Union Bacterial Resistance Surveillance Network across Sichuan from 2015 to 2018, so as to provide reference for clinical rational drug use and management of drug-resistant bacteria in Sichuan.MethodsA total of 18 023 strains of staphylococci were isolated from 9 hospitals of Whire Union Bacterial Resistance Surveillance Network for four years (2015-2018). Drug susceptibility test was carried out by disk diffusion method or automated instrument method. The data were statistically analyzed by WHONET 5.6 according to CLSI 2016 standard.ResultsThe 18 023 strains of staphylococci included 10 865 (60.28%) Staphylococcus aureus and 7 158 (39.72%) coagulase negative staphylococci. No strains resistant to vancomycin and linezolid were found. The detection rates of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci were 25.10% (2 727/ 10 865) and 75.60% (5 411/7 158), respectively. The sensitivity of methicillin-resistant staphylococci to most antibiotics was significantly lower than that of methicillin-sensitive strains (P<0.05). The susceptibility rate of staphylococci to some antibiotics was significantly different from 2015 to 2018(P<0.05). The susceptibility rates of Staphylococcus aureus from different samples to rifampicin, moxifloxacin, ciprofloxacin, levofloxacin, oxacillin and erythromycin were significantly different (P<0.05). The susceptibility rates of Staphylococcus aureus from different departments in different samples of sulfamethoxazole, rifampicin, moxifloxacin, ciprofloxacin, levofloxacin, oxacillin, gentamicin, tetracycline, clindamycin and erythromycin were significantly different (P<0.05).ConclusionsThe susceptibility of strains isolated from different periods, different specimens and departments to the same antimicrobial agents varies greatly. For the infection of staphylococci, we should use drugs under the guidance of drug susceptibility according to the source of samples, which can avoid the abuse of beta-lactam drugs. Strengthening the monitoring and control of drug-resistant bacteria can prevent or reduce the spread of drug-resistant bacteria.
Objective To study the influence of brominated furanones on the biofilm (BF) formation of Staphylococcus epidermidis (SE) on polyvinyl chloride(PVC) materials, and provide new ideas for the research of surface modification of materials and clinical treatment of biomaterial centered infection. Methods We chose three kinds of brominated furanone with representative chemical structure for our research which were respectively 3,4dibromo-5-hydroxy2 (5H) -furanone (Mucobromic acid) in the first furanone group, 4-bromo-5(4-methoxyphenyl)3(methylamino)2(5H)furanone in the second furanone group, and 3,4dibromo-5,5-bis(4-methylphenyl)2(5H)-furanone in the third furanone group. The PVC material soaked with 75% ethanol for 5minutes was classified as the control group. The surface coating of the PVC materials in the four groups all underwent modification respectively and then they were cocultivated with staphylococcus epidermidis together. Confocal laser scanning microscope(CLSM) was adopted to detect the thickness of bacterium BF and bacterium community quantity unit area on PVC materials and scanning electron microscope(SEM) was used to observe surface structure of SE, BF formation at 6 h, 12 h, 18 h and 24 h respectively. Results The results of CLSM showed that, compared with the control group, SE bacterium community quantity unit area and the thickness of bacterium BF on the PVC material surface in the second furanone group were obviously smaller (Plt;0.05). SE bacterium community quantity unit area and thickness of bacterium BF on PVC material surface in the first and the third furanone groups had no significant difference (Pgt;0.05). The result of SEM showed that, the quantity of SE bacterium community unit area on PVC material surface in the second furanone group were smaller than that of the control group at 6 hours. The biofilm structure on PVC material surface in the control group was formed at 18 hours, but there were no mature biofilm structure on PVC material surface in the second furanone group at 18 hours. Conclusion The impact of different brominated furanone on SE biofilm formation on the surface of PVC materials is different. The second kind of furanone can inhibit the quantity of SE bacterium community unit area and SE biofilm formation on the surface of PVC materials.