Objective To survey and analyze the drug resistance of clinical isolated strains of bloodborne infectious staphylococci, in order to provide references for clinical diagnosis of aureus septicemia and rational use of antimicrobial agents. Methods We retrospectively studied the clinical data of 496 patients with staphylococcal septicemia confirmed by blood culture between June 2008 and May 2015 in West China Hospital of Sichuan University. The microbiological characteristics of the disease were analyzed. Results In the included 496 cases, there were 216 (43.55%) cases of coagulase-positive Staphylococcus (CPS) septicemia and 280 (56.45%) of coagulase-negative Staphylococcus (CNS) septicemia; 85 (17.14%) cases were caused by community infection, while the other 411 (82.86%) resulted from hospital infection. The drug resistance rate of CPS and CNS toward oxacillin was respectively 27.78% (60/216) and 87.50% (245/280), with a significant difference (P < 0.05). In al l the clinical isolated strains of CPS, the drug resistance rate of community infected strains and hospital infected strains toward oxacillin was respectively 9.67% (6/62) and 35.06% (54/154), with a significant difference (P < 0.05). For the clinical isolated strains of CNS, the drug resistance rate of community infected strains and hospital infected strains toward oxacillin was respectively 69.57% (16/23) and 89.11% (229/257), also with a significant difference (P < 0.05). Conclusions The drug resistance of hospital infected staphylococcal strains is stronger than community infected strains. The CNS strains are more drug-resistant than CPS strains.
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 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 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 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 Mesh infection may occur after incisional hernia repair using prosthetic mesh. Preparation of antibiotics-bonded meshes to prevent infection is one of the solutions. To evaluate the anti-infection effect of polypropylene mesh bonded norvancomycin slow-release microsphere by preparing the rat model of incisional hernia repair contaminatedwith Staphylococcus aureus. Methods The norvancomycin slow-release microspheres were prepared by emulsion and solvent evaporation method and they were bonded to polypropylene mesh (50 mg/mesh). The appearance of the microspheres was observed using scanning electronic microscope (SEM). The content of norvancomycin in microspheres and the release rate of the norvancomycin in norvancomycin-bonded polypropylene mesh were detected using high performance l iquid chromatography method. The rat models of incisional hernia were developed in 40 healthy Sprague Dawley rats, aged 10-11 weeks and weighing 200-250 g. The rats were divided randomly into the experimental group (norvancomycin-bonded polypropylene mesh repair, n=20) and the control group (polypropylene mesh repair, n=20). And then the mesh was contaminated with Staphylococcus aureus. The wound heal ing was observed after operation. At 3 weeks after operation, the mesh and the tissue around the mesh were harvested to perform histological observation and to classify the inflammatory reaction degree. Results The norvancomycin microsphere had integrated appearance and smooth surface with uniform particle diameter, 64% of particlediameter at 60 to 100 μm, and the loading-capacity of norvancomycin was 19.79%. The norvancomycin-bonded polypropylene patch had well-distributed surface and the loading-capacity of norvancomycin was (7.90 ± 0.85) mg/cm2. The release time of norvancomycin in vitro could last above 28 days and the accumulative release rate was 72.6%. The rats of 2 groups all survived to experiment completion. Wound infection occurred in 2 rats of the experimental group (10%) and 20 rats of the control group (100%), showing significant difference (χ2=32.727 3, P=0.000 0). The inflammatory reaction in experimental group was not obvious, grade I in 16 rats and grade II in 4 rats, and numerous inflammatory cell infiltration occurred in the control group, grade II in 3 rats and grade III in 17 rats, showing significant difference (Z=32.314, P=0.000). Conclusion The polypropylene mesh bonded norvancomycin slow-release microsphere has definite anti-infection effect in rat model of incisional hernia repair contaminated by Staphylococcus aureus.
ObjectiveTo summarize the clinical features and experience of Methicillin-resistant Staphylococcus aureus (MRSA)-associated enteritis. MethodsClinical data of 21 patients with MRSA-associated enteritis who were treated in our hospital from Jan. 2003 to May. 2015 were analyzed retrospectively. ResultsAfter diagnosed or suspected of MRSA-associated enteritis, the 21 patients received a drug therapy with vancomycin instead of other antibiotic, 3 patients (14.3%) who failed to get satisfactory symptom relief received a plus therapy with biapenem; 13 patients (61.9%) received treatment which plus drugs such as Bacillus licheniformis capsules or combining Bifidobacterium to regulate intestinal microflora. Severe complications, such as intestinal fistula (8 patients, 38.1%）, toxic shock (16 patients, 76.2%）, organ system failure (14 patients, 66.7%) occurred in 17 patients (80.9%) of the 21 patients when 2-7 days (mean of 4.7 days) after diarrhea. Among 21 patients received therapy, 7 patients (33.3%) were cured and 2 patients (9.5%) were improved, whereas 11 patients died, with a total mortality of 52.4%, another 1 patient was lost to follow up (4.8%). There were 8 patients who were followed-up for 1-12 months (the median time was 3.1-month). During the followed-up period, 2 of them died and others stayed alive without occurrence. ConclusionAlthough uncommon, MRSA-associated enteritis progressed rapidly, with many complications and high mortality rate. Early diagnosis and timely targeted treatment restoring the balance of gastrointestinal microecology are the key to decrease its mortality.
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 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 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.