OBJECTIVE: To investigate the ability of repairing bone defect with the compound of coralline hydroxyapatite porous (CHAP), fibrin sealant(FS) and staphylococcus aureus injection (SAI), and the feasibility to use the compounds as bone substitute material. METHODS: The animal model of bone defect was made on the bilateral radius of 54 New Zealand white rabbits, which were randomly divided into the experimental group(the defect was repaired with CHAP-FS-SAI), control group(with autograft) and blank control group(the defect was left unrepaired) with 18 rabbits in each group. The ability of bone defect repair was evaluated by gross observation, histopathological study, X-ray and biomechanical analysis 2, 4, 8 and 12 weeks after repair. RESULTS: (1) In the 2nd week, tight fibro-connection could be found between the implant and fracture site and there were many fibroblasts and capillary proliferation with many chondrocytes around CHAP in the experimental group, while only a few callus formed, and chondrocytes, osteoblast and osteoclast existed in the control group. (2) In experimental group and control group, a large quantity of callus was found 4 and 8 weeks; ossification of chondrocytes with weave bone formation were found 4 weeks and many osteocytes and weave bones and laminar bones were found 8 weeks. (3) In the 12th week, the complete ossification of implant with well bone remodeling, a large number of mature osteocytes and laminar were found in experimental group and control group, and CHAP still existed in the experimental group; the defect area filled with fibro-scar tissue and only many fibroblasts could be seen in blank control group. (4) X-ray findings were the following: In experimental and control groups, callus formation could be seen 2 weeks postoperatively, more callus formed 4 weeks, the bone defect area disappeared and CHAP scattered in the callus 8 weeks; the fracture line disappeared and medullary cavity became united (in control group); and in the 12th week, the cortex became continuous, the medullary cavity became united, and remodeling completed, while bone defect was not still united in blank control group. The maximal torque and torsional stiffness in the experimental group is higher than those in the control group 2 weeks (P lt; 0.05), but there was no significant difference (P gt; 0.05) between the two groups 4, 8, 12 weeks after repair. CONCLUSION: The compound of CHAP-FS-SAI has good biological compatibility, and it can be used for one kind of bone substitute material to repair the bone defect.
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 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.
ObjectiveTo investigate the correlation between infection and capsular contracture by observing the effect of infection on the formation of the surrounding capsule after breast implants. MethodsThree healthy adult female Diannan small-ear pigs underwent augmentation mammaplasty using miniature implants, which were randomly divided into group A (12 nipples), group B (10 nipples), and group C (12 nipples). Staphylococcus epidermidis (SE ATCC12228 and SE RP62A, 1.2×105 CFU/mL) was inoculated into the periprosthetics of groups B and C, and sterile PBS in group A before breast implants. Then the silica gel prosthesis was put, total 34 implants in 3 groups. After 13 weeks, the capsule was harvested to measure the capsular tension and weight. HE staining was used to observe the structure characteristics of the capsule and to measure the capsule thickness, Van-Gieson (VG) staining to observe the capsule collagen characteristics, and α-smooth muscle actin (α-SMA) immunocytochemistry staining to observe myofibroblasts in capsule. ResultsPrimary healing of incision was obtained, and 3 small-ear pigs showed stable life indication. The complete fibrous capsule was observed after 13 weeks in 3 groups. Capsule tension showed no significant difference among 3 groups (P>0.05). Capsule weight was significantly greater in group C than in groups A and B (P<0.05). HE staining showed that capsule structure of the 3 groups was similar with obvious dense layer and loose layer, and the capsule thickness was also significantly greater in group C than in groups A and B (P<0.05), but no significant difference was found between groups A and B (P>0.05). VG staining showed that collagenous fiber in the capsule were more compact in group C than in groups A and B. The α-SMA immunocytochemistry staining indicated the myofibroblasts in capsule were the most in group C. ConclusionInfection after breast implants has obvious impacts on the formation of the capsule, and there was a causal link between infection and capsular contracture.
Objective To investigate the incidence rate, molecular epidemiology and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) infection. Methods A total of 119 Staphylococcus aureus strains isolated from January 2016 to December 2020 in general surgery of this hospital were collected retrospectively and divided into MRSA group and methicillin-sensitive Staphylococcus aureus group according to whether or not resistant to oxacillin. The clinical data of all patients infected with Staphylococcus aureus and drug sensitivity of Staphylococcus aureus were collected. Molecular typing was performed by multilocus sequence typing (MLST), resistance gene, virulence gene and biofilm gene were detected by polymerase chain reaction (PCR) method, and a case-control study was used to identify risk factors for MRSA infection. ResultsThe detection rate of MRSA was 57.98% (69/119), mainly was from pus specimens (80.67%, 96/119). The results of MLST showed that the dominant clone types were ST88 (37.68%, 26/69), ST951 (27.54%, 19/69) and ST59 (18.84%, 13/69). The results of PCR showed that the detection rates of mecA, mecC, Aac (6′ )/Aph (2′ ′ ), Aph (3)-Ⅲ, ant (4′ )- Ⅰ a, tetM, qnrA, panton-valentine leukocidin, fibronectin-binding protein A, staphylococcal enterotoxin A, staphylococcal enterotoxin B, α-hemolysins, intracellular adhesion A, staphylococcal accessory regulators A, and fibronectin-binding protein B in 69 strains of MRSA were 100%, 0.00%, 27.54%, 34.78%, 18.84%, 14.49%, 1.45%, 8.70%, 98.55%, 11.59%, 91.30%, 94.20%, 92.75%, 97.10% and 86.96%, respectively. Multivariate analysis showed that hospital transfer, wound infection, catheter related infection, drainage tube and history of cephalosporin using were risk factors for MRSA infection. ConclusionsThe detection rate of MRSA in general surgery of this hospital is high. ST88 is the most common clone type. The carrying rates of resistant-, virulence- and biofilm-related genes are high. Hospital transfer, wound infection, drainage tube, history of cephalosporin using etc. are high risk factors for MRSA infection. It is advised that invasive operation should be reduced, antibiotics should be used rationally, hand hygiene should be paid attention to, environmental sanitation disinfection should be carried out regularly, and the monitoring of MRSA bacteria should be strengthened, so as to reduce and control the infection and spread of MRSA.
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 analyze the pathogenic bacteria distribution, structure and characteristics of drug resistance in patients with acute stroke complicated with pulmonary infection, in order to provide reference for the prevention of hospital infection and rational use of antimicrobial agents. MethodsA total of 864 clinical specimens of acute stroke complicated with pulmonary infection were chosen for study between January 2012 and December 2014. Separation and cultivation were done in accordance with the operation procedures regulated by the Ministry of Health. Drug sensitivity examination was done by Kirby-Bauer (k-b). Super-extensive spectrum β lactamase (ESBL) and methicillin resistant staphylococcus aureus (MRSA) were detected to analyze the bacterial species and resistance transition. ResultsA total of 864 samples were cultivated, in which G-bacteria accounted for 61.2%. The main pathogenic bacteria was Klebsiella pneumoniae bacteria, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanmii and Staphylococcus aureus. Imipenem had high antimicrobial activity to G-bacilli, especially to Escherichia coli and Klebsiella pneumoniae bacteria. Linezolid, vancomycin and teicoplanin had high antibacterial activity to staphylococcus aureus. Vancomycin resistant Staphylococcus aureus was not found. Ciprofloxacin had high antibacterial activity to Pseudomonas aeruginosa, while imipenem had low antibacterial activity to Pseudomonas aeruginosa. Amikacin had high antibacterial activity to acinetobacter. ConclusionG-bacilli are predominant in acute stroke complicated with pulmonary infection. ESBLs and MRSA detection rate is high, and we should pay attention to the rational use of antibiotics to reduce drug resistance.
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 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 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.