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.
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.
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 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 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.
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.
ObjectiveTo investigate the appropriate concentration of methicillin-resistant Staphylococcus aureus (MRSA) in establishing chronic femoral osteomyelitis model in rabbits.MethodsForty-eight adult New Zealand white rabbits were randomly divided into 6 groups with 8 rabbits in each group. Animals in groups B, C, D, E, and F were injected 1×109, 1×108, 1×107, 1×106, 1×105 CFU/mL MRSA on the location of 2 cm of the femoral supracondyle, respectively, and group A was injected with aseptic saline as a control. The general observation were performed at 4 weeks after operation, and the wound secretions were taken for bacteriological examination. The serum C-reactive protein content was detected at preoperation and 2 weeks and 4 weeks after operation. The X-ray, CT scan, and Norden imaging scoring were performed at 4 weeks after operation. At 4 weeks after operation, the animals were sacrificed, and the specimens were observed and evaluated by general scores; and the HE staining and histological score were also performed.ResultsFive rabbits died of severe infection in group B, 2 died in group C, and no rabbit died in groups D, E, and F. General observation showed that the incision healed without soft tissue swelling in group A; most animals had visible incision swelling and sinus formation, femoral thickening, bone destruction, and damage decreased with the decreasing of the concentration of liquid bacterial in groups B-D; the infection signs were seen in groups E and F, and the degree of infection were less than that of group D. Bacteriological examination showed that fistula formation animal in groups B, C, D, and E were cultured with positive results, and with the decrease of concentration, the number of animal fistula formation decreased gradually; and bacteriological culture did not be performed in group F because of no sinus formation. There was no significant difference in the content of C-reactive protein between groups before operation (P>0.05). The contents of C-reactive protein in groups B-F were significantly higher than those in group A at 2 and 4 weeks after operation (P<0.05). At 4 weeks after operation, the content of C-reactive protein was in the order of groups B, C, D, E, F, and A in turn from high to low, showing significant differences between groups (P<0.05). Imaging examination showed that there was no soft tissue swelling and bone destruction in group A; bone destruction, massive sequestrum formation, and soft tissue swelling were found in groups B and C; bone destruction was observed in groups D and E, and the degree of sequestrum formation was not as good as that in group C; and there was a small amount of bone infection in group F. The Norden scores in groups B-F were significantly higher than that in group A, and in groups B and C than those in groups D, E, and F, and in groups D and E than that in group F (P<0.05); there was no significant difference between groups B and C, and between groups D and E (P>0.05). The specimens general observation scores in groups B-F were significantly higher than that in group A, while in groups B and C than those in groups D, E, and F (P<0.05); there was no significant difference between groups D, E, and F (P>0.05). HE staining showed that the structure of bone trabecula in group A was clear and the structure was arranged neatly; in groups B-F, trabecular bone destruction and inflammatory cell infiltration were seen and the degree gradually decreased. The histological scores in groups B-F were significantly higher than that in group A, and in group B than those in groups C-F, in groups C and D than that in group F (P<0.05); there was no significant difference between groups C, D, and E, and between groups E and F (P>0.05).ConclusionThe optimal MRSA concentration of rabbit model of chronic osteomyelitis of femur is between 1×106 and 1×107 CFU/mL.
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.