医源性表皮葡萄球菌胞间黏附素基因操纵子在聚氯乙烯材料表面细菌生物膜形成中的作用研究

目的表皮葡萄球菌胞间黏附素基因(intercellar adhesion,ica)是细菌聚集的关键因子,通过分析医源性表皮葡萄球菌生物膜基因型,探讨ica操纵子在聚氯乙烯(polyvinyl chloride,PVC)材料表面细菌生物膜形成中的作用。方法取56株医源性表皮葡萄球菌临床分离株,应用PCR法、基因测序技术检测细菌生物膜形成相关基因,包括16S rRNA、自溶素(autolysin,atlE)、纤维蛋白原结合蛋白(fi brinogen binding protein,fbe)以及ica。取医源性表皮葡萄球菌制备浓度为1×105cfu/mL的细菌悬液,并根据目的基因检测结果,分别以icaADB、atlE、fbe阳性基因型(ica操纵子阳性组)以及icaADB阴性而atlE、fbe阳性基因型(ica操纵子阴性组)与PVC材料培养。于6、12、18、24、30h各取2个PVC材料,进行激光共聚焦显微镜及扫描电镜观察,测量单位视野细菌群落数量及形成的细菌生物膜厚度。结果目的基因检测示,医源性表皮葡萄球菌16S rRNA阳性率为100%(56/56);icaADB、atlE、fbe阳性基因型菌株占57.1%(32/56);icaADB阴性而atlE、fbe阳性基因型菌株占37.5%(21/56)。测序结果示,目的基因16S rRNA、atlE、fbe、icaADB扩增产物序列分别与GenBank中基因序列相符。随时间延长,ica操纵子阴性组的PVC材料表面无明显细菌生物膜形成;ica操纵子阳性组的PVC材料表面细菌群落数量逐渐增多,细菌生物膜体积逐渐增大,24h时可见成熟的细菌生物膜结构,30h时细菌生物膜体积趋于稳定。培养各时间点ica操纵子阳性组PVC材料表面单位视野细菌群落数量(F=435.987,P=0.000)及细菌生物膜厚度(F=6714.395,P=0.000)明显高于ica操纵子阴性组,差异均有统计学意义。结论医源性表皮葡萄球菌可以分为ica操纵子阴性和ica操纵子阳性两类细菌;ica操纵子可以增加PVC材料表面细菌生物膜的形成能力、细菌群落数量及细菌生物膜厚度,在PVC材料表面细菌生物膜形成中具有重要作用。     

溴代呋喃酮对聚氯乙烯材料表面大肠杆菌生物膜形成的影响

目的探讨不同溴代呋喃酮对聚氯乙烯(polyvinyl chloride,PVC)材料表面大肠杆菌生物膜形成的影响,为生物材料表面改性研究及临床生物材料植入感染的防治提供新思路。方法选用具有化学结构代表性的3种溴代呋喃酮,溴代呋喃酮1:3,4-二溴基-5-羟基-呋喃酮,溴代呋喃酮2:4-溴-5-(4-甲氧基苯基)-3-(甲氨基)-呋喃酮,溴代呋喃酮3:3,4-二溴基-5,5-二甲苯基-2(5H)-呋喃酮,分别对PVC材料片(1cm×1cm)进行表面涂层改性,将改性后的PVC材料片与大肠杆菌共同培养;将PVC材料片用75%乙醇浸泡5min后与大肠杆菌共同培养作为对照组。分别于培养6、12、18、24h,采用激光共聚焦显微镜动态观测PVC材料表面单位面积细菌群落数及细菌群落厚度,扫描电镜观察PVC材料表面细菌生物膜表面结构。结果激光共聚焦显微镜观测显示,各时间点溴代呋喃酮3组PVC材料表面单位面积细菌群落数以及细菌群落厚度均小于对照组,差异有统计学意义(P0.05);而溴代呋喃酮1组和溴代呋喃酮2组与对照组比较差异均无统计学意义(P0.05)。扫描电镜观察示,与对照组比较,溴代呋喃酮3组培养后6h PVC材料表面细菌群落附着数量较少;18h时对照组及溴代呋喃酮1组和溴代呋喃酮2组PVC材料表面细菌生物膜结构已初步形成,而溴代呋喃酮3组PVC材料表面无明显细菌生物膜结构形成。结论不同溴代呋喃酮对PVC材料表面大肠杆菌生物膜形成的影响不同,3,4-二溴基-5,5-二甲苯基-2(5H)-呋喃酮可抑制PVC材料表面单位面积大肠杆菌群落数和细菌群落厚度形成。     

金属蛋白酶对涤纶材料表面金黄色葡萄球菌生物膜形成的影响

目的观察不同浓度下金属蛋白酶(A ur)对金黄色葡萄球菌在涤纶材料表面细菌生物膜形成的影响。方法90片涤纶片按随机数字表法分成3组,每组30片。3组均将涤纶片放入有金黄色葡萄球菌液(105CFU/m l)的无菌瓶中,A组再加入含400ng/m l浓度的A ur,B组再加入含80ng/m l浓度的A ur。3组均置入电热恒温水浴箱(98.6°F)中培育,分别于培育后6h、16h、24h、30h和48h用扫描电子显微镜(SEM),激光共聚焦扫描显微镜(CLSM)观察细菌生物膜的厚度和单位面积内细菌生物膜群落数量。结果对照组涤纶片细菌生物膜的厚度和群落数呈时间依赖性明显增加,A组涤纶片金黄色葡萄球菌生物膜的厚度和群落数在各时间点均明显低于B组和对照组(P<0.05),B组生物膜的厚度和群落数在各时间点亦明显低于对照组(P<0.05)。结论A ur对金黄色葡萄球菌在涤纶片上细菌生物膜的形成有抑制作用,其抑制作用的强度与A ur剂量呈正相关。     

人工关节假体不同材料及材料表面粗糙度对表皮葡萄球菌粘附能力的影响

目的探讨人工关节假体材料及表面粗糙度对表皮葡萄球菌粘附能力的影响。方法制作超高分子聚乙烯、钛合金和钴铬钼合金试样，表皮葡萄球菌经FITC标记，人工关节材料试样消毒后接种FITC标记的表皮葡萄球菌，试样表面分为光滑表面和粗糙表面，每组各6个试样，将含有细菌和试样的24孔板在37℃下孵育30min后，用荧光显微镜观察，试样用扫描电镜观察。结果对于光滑的人工关节常用材料表面，表皮葡萄球菌对超高分子聚乙烯（UHMWPE）的粘附能力显著高于钛合金和钴铬钼合金（P〈0．001），对钴铬钼合金的粘附能力要高于钛合金（P〈0．05）；粗糙的超高分子聚乙烯和钴铬钼合金表面比光滑的表面更易引起表皮葡萄球菌的粘附（P〈0．01），而细菌对粗糙钛合金的粘附仅轻微高于光滑钛合金（P〉0．05）。荧光照相观察及扫描电镜观察显示细菌在粗糙表面的划痕内聚集粘附。结论本研究结果表明细菌对人工关节材料表面的粘附能力不但取决于细菌本身，也和材料性质和表面粗糙度有关。     

表皮葡萄球菌生物膜形成相关基因在表皮葡萄球菌和白假丝酵母菌混合生物膜形成中的作用研究

目的探讨表皮葡萄球菌生物膜形成相关基因——胞间黏附素A(intercellular adhesion A,ica A)基因、纤维蛋白原结合蛋白(fibrinogen binding protein,fbe)基因、聚集相关蛋白(accumulation-associated protein,aap)基因在表皮葡萄球菌和白假丝酵母菌混合生物膜形成中的作用。方法实验分为3组,用表皮葡萄球菌标准株ATCC35984(表葡组)及白假丝酵母菌标准株ATCC10231(白念组)分别培养及混合培养(混合组),建立表皮葡萄球菌、白假丝酵母菌及二者混合生长的体外生物膜模型。于培养2、4、6、8、12、24、48、72 h,采用结晶紫染色法半定量检测生物膜形成能力,二甲氧唑黄[(2,3 bis(2-methoxy-4-nitro-5-sulfophenyl)5〔(phenylamino)Carbonyl〕2H-tetrazolium hydroxide assay,XTT]比色法评价生物膜体外生长动力学;24、72 h扫描电镜观察生物膜超微结构。荧光定量PCR分析培养72 h表葡组及混合组ica A、fbe、aap基因表达情况。结果结晶紫染色法生物膜半定量检测示,混合组和表葡组均在培养12 h生物膜明显增厚,72 h混合组超过表葡组,组间比较除72 h外,其余各时间点两组比较差异均有统计学意义(P0.05);白念组12 h出现生物膜的生长,在整个培养周期白念组生物膜厚度均低于混合组(P0.05)。XTT比色法生长动力学检测示,混合组整体生长速度快于白念组,且48 h后超过表葡组;混合组与表葡组除12 h差异有统计学意义(P0.05)外,其余各时间点比较差异均无统计学意义(P0.05);混合组培养2、4 h时A值低于白念组,但比较差异无统计学意义(P0.05);6 h后各时间点A值均显著高于白念组(P0.05)。扫描电镜观察示,随培养时间延长各组均形成结构复杂、成熟的生物膜。培养72 h荧光定量PCR检测示,与表葡组相比,混合组fbe、ica A、aap基因表达量分别增高1.93、1.52、1.46倍,差异均有统计学意义(P0.05)。结论表皮葡萄球菌和白假丝酵母菌混合生长能形成比单一微生物结构更复杂的混合生物膜;混合生物膜较单一微生物生物膜更厚,可能与表皮葡萄球菌ica A、aap、fbe基因表达增加有关。     

表皮葡萄球菌生物膜蛋白在假体周围感染诊断中的应用

目的探讨表皮葡萄球菌生物膜蛋白作为抗原诊断假体周围感染的临床价值。方法制作表皮葡萄球菌感染的假体周围感染动物模型，并收集临床假体周围感染患者的血清。以从表皮葡萄球菌444生物膜中提取的蛋白为抗原，用ELISA方法检测感染动物和临床假体周围感染患者的血清中IgG水平。蛋白印迹杂交寻找特异性抗原蛋白。结果感染动物和感染患者血清中的IgG水平明显高于各自对照组，Western—blot结果显示，表皮葡萄球菌生物膜蛋白大小在15KD-37KD之间具有良好的抗原性。结论表皮葡萄球菌生物膜蛋白成份具有良好的抗原性，是引起机体免疫的重要成份。进一步纯化这些蛋白组分并作为诊断假体周围表皮葡萄球菌感染的抗原，具有重要的临床价值。     

聚氯乙烯材料表面白色念珠菌-表皮葡萄球菌混合生物膜结构观察

目的建立聚氯乙烯(polyvinyl chloride,PVC)材料表面白色念珠菌-表皮葡萄球菌混合生物膜体外模型,观察混合生物膜的形成与微观结构。方法取表皮葡萄球菌(ATCC35984)与白色念珠菌(ATCC10231),分别制备浓度为1×106 CFU/m L的悬液并混合后,与直径0.5 cm PVC膜在胰蛋白胨大豆肉汤(tryptic soy broth,TSB)培养基中共培养形成混合生物膜(实验组)。培养2、6、12、24、48、72 h时取PVC膜,激光共聚焦显微镜检测生物膜厚度、单位视野菌落数,并于48 h时测量生物膜内活菌百分比、三维重建PVC膜表面生物膜图像;扫描电镜观察各时间点混合生物膜结构。以单纯PVC膜置于TSB培养基中培养作为对照组。结果对照组各时间点PVC材料表面无病原菌黏附。实验组激光共聚焦显微镜观察示,培养6 h时可见菌落及生物膜形成,随时间延长均逐渐增加,24 h时菌落达高峰,48 h时生物膜厚度达峰值。实验组PVC膜表面菌落数比较,2、6、24 h间以及2、6 h与48、72 h间比较差异有统计学意义(P0.05),24、48、72 h间比较差异无统计学意义(P0.05);生物膜厚度除48、72 h间比较差异无统计学意义外(P0.05),其余各时间点间比较差异均有统计学意义(P0.05)。培养48 h时,混合生物膜外层活菌百分比明显高于内层及中间层(P0.05)。三维重建显示混合生物膜表面凹凸不平,突起部分活菌数量较多。扫描电镜观察示,实验组随培养时间增加,PVC膜表面白色念珠菌由孢子状逐渐伸长出现假丝状及菌丝状,表皮葡萄球菌黏附于白色念珠菌周围,逐渐形成复杂的多层次网状混合生物膜。结论白色念珠菌-葡萄球菌混合培养可在PVC材料表面形成结构复杂的混合生物膜,激光共聚焦显微镜、扫描电镜与三维图像重建技术的结合是研究混合生物膜的理想方法。     

新型表皮细胞生物膜移植物的研究

目的将人表皮细胞接种到生物膜上构建一种新的表皮替代物———表皮细胞生物膜移植物（ＭＣＧ）。方法新鲜和冷冻保存的ＭＣＧ移植到裸鼠全厚皮肤缺损创面,以单纯无接种细胞的生物膜作为对照。并行组织学、免疫组织化学及电镜观察。结果表皮细胞可以生物膜为载体在体外进行培养,达６０％～７０％亚饱和状态时,表皮细胞生物膜可一起移植到创面,在创面上表皮细胞继续增殖分化,形成一层新生表皮。结论表皮细胞生物膜移植物可作为一种新型表皮替代物修复皮肤缺损     

急性期骨科钛合金板表面金黄色葡萄球菌细菌生物膜的结构演变

目的 采用激光共聚焦显微镜及扫描电子显微镜技术动态观察急性期(4周)内不同时相点钛合金板表面金黄色葡萄球菌的生物膜结构。方法 使用骨科钛合金板培养体外金黄色葡萄球菌形成不同时相点的生物膜,在培养1周、2周、3周、4周后取出钛合金板,用异硫氰酸荧光素标记的刀豆蛋白和碘化丙啶染料将不同时相点的生物膜染色,染色后使用激光共聚焦显微镜和扫描电子显微镜观察细菌生物膜的形态结构。结果 得到不同时相点的生物膜的激光共聚焦图像及显微电镜图像,1周时细菌胞外少量多糖聚合物形成,内部空间结构杂乱无序,表明形成了早期生物膜。随着培养时间的延长细菌胞外多糖聚合物逐渐增多,内部间隙及孔道相互交通,空间结构逐渐复杂。到4周时大量胞外多糖聚合物形成,膜内结构进一步完善,表明形成成熟的生物膜。各时相点内活菌数目,差异无统计学意义(P>0.05);各时相点间生物膜厚度,差异有统计学意义(P<0.05)。结论 金黄色葡萄球菌细菌生物膜有早期和晚期区别,从早期生物膜到晚期成熟生物膜是一个动态演变过程,表现为细菌生物膜胞外多糖聚合物及内部空间结构的成熟稳定。     

宁泌泰胶囊对葡萄球菌和大肠杆菌及其生物膜体外形成抑制作用的研究

目的:评估宁泌泰胶囊对葡萄球菌和大肠杆菌增殖以及体外培养生物膜的抑制作用. 方法:使用梯度稀释法检测表皮葡萄球菌(1457),金黄色葡萄球菌(NCTC8325-4、Newman、MU50),大肠埃希菌(ATCC25922、CFT073)对宁泌泰的最低抑菌浓度(MIC);使用扫描电镜,观察不同浓度的宁泌泰对表皮葡萄球菌1...     

Berberine inhibits Staphylococcus Epidermidis adhesion and biofilm formation on the surface of titanium alloy

Biofilm formed by Staphylococcus epidermidis (S. epidermidis) is a common cause of periprosthetic infection. Recently, we have discovered that berberine is bacteriostatic for S. epidermidis. The purpose of the present study was to examine the effect of berberine on S. epidermidis adhesion and biofilm formation on the surface of titanium alloy, which is a popular material for orthopedic joint prostheses. Three strains of S. epidermidis (ATCC 35984, ATCC 12228, and SE 243) were used for in vitro experiment. Direct colony counting showed that berberine significantly inhibited S. epidermidis adhesion on the titanium alloy disk in 2 h at the concentration of 45 µg/mL. When examined with crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy, we found that higher concentrations (>30 µg/mL) of berberine effectively prevented the formation of S. epidermidis biofilm on the surface of the titanium disk in 24 h. These findings suggest that berberine is a potential agent for the treatment of periprosthetic infection. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1487–1492, 2009     

Influence on Blood of Plasticized Polyvinyl Chloride: Significance of the Plasticizer

An investigation has been made of the significance of the level of the plasticizer di-(2-ethylhexyl)-phthalate at the surface of plasticized polyvinyl chloride for interactions with blood components. Plasticized polyvinyl chloride before and after treatment with methanol to reduce the plasticizer surface level was assessed in terms of fibrinogen and albumin adsorption with unplasticized polyvinyl chloride acting as a control. As the plasticizer surface level decreased, fibrinogen adsorption decreased almost linearly while albumin adsorption increased initially before decreasing slightly. The investigation indicates that reduction in the amount of plasticizer at the surface improves the blood compatibility of plasticized polyvinyl chloride, and the influence on blood is due primarily to the plasticizer rather than the polyvinyl chloride itself.     

The Influence of Maggot Excretions on PAO1 Biofilm Formation on Different Biomaterials

Biofilm formation in wounds and on biomaterials is increasingly recognized as a problem. It therefore is important to focus on new strategies for eradicating severe biofilm-associated infections. The beneficial effects of maggots (Lucilia sericata) in wounds have been known for centuries. We hypothesized sterile maggot excretions and secretions (ES) could prevent, inhibit, and break down biofilms of Pseudomonas aeruginosa (PAO1) on different biomaterials. Therefore, we investigated biofilm formation on polyethylene, titanium, and stainless steel. Furthermore, we compared the biofilm reduction capacity of Instar-1 and Instar-3 maggot ES and tested the temperature tolerance of ES. After biofilms formed in M63 nutrient medium on comb-forming models of the biomaterials, ES solutions in phosphate-buffered saline or M63 were added in different concentrations. PAO1 biofilms adhered tightly to polyethylene and titanium but weakly to stainless steel. Maggot ES prevent and inhibit PAO1 biofilm formation and even break down existing biofilms. ES still had considerable biofilm reduction properties after storage at room temperature for 1 month. ES from Instar-3 maggots were more effective than ES from Instar-1 maggots. These results may be relevant to patient care as biofilms complicate the treatment of infections associated with orthopaedic implants. One or more of the authors (GC, GNJ) have received funding from an unrestricted research grant of Kinetic Concepts Inc, Europe Holding BV, Amstelveen, The Netherlands.     

血肿在硬膜外瘢痕粘连形成中作用的探讨

为减少术后硬膜外神经根瘢痕粘连，研究血肿在瘢痕形成中的作用。方法兔２５只行Ｌ３、Ｌ５、Ｌ７间隔三处的椎板切除，术后硬膜外置管生理盐水冲洗７２小时，另二处分别为单纯引流和空白对照。术后３天、２、４、８、１２周取材，观察硬膜外瘢痕形成过程。     

Biofilm dispersal of community‐associated methicillin‐resistant Staphylococcus aureus on orthopedic implant material

Orthopedic implant‐related bacterial infections are associated with high morbidity that may lead to limb amputation and exert significant financial burden on the healthcare system. Staphylococcus aureus is a dominant cause of these infections, and increased incidence of community‐associated methicillin‐resistant S. aureus (CA‐MRSA) is being reported. The ability of S. aureus to attach to the foreign body surface and develop a biofilm is an important determinant of resistance to antibiotic prophylaxis. To gain insight on CA‐MRSA biofilm properties, USA300 biofilm maturation and dispersal was examined, and these biofilms were found to exhibit pronounced, quorum‐sensing mediated dispersal from a glass surface. For comparison of biofilm maturation on different surface chemistries, USA300 biofilm growth was examined on glass, polycarbonate, and titanium, and minimal differences were apparent in thickness, total biomass, and substratum coverage. Importantly, USA300 biofilms grown on titanium possessed a functional dispersal mechanism, and the dispersed cells regained susceptibility to rifampicin and levofloxacin treatment. The titanium biofilms were also sensitive to proteinase K and DNaseI, suggesting the matrix is composed of proteinaceous material and extracellular DNA. These studies provide new insights on the properties of CA‐MRSA biofilms on implant materials, and indicate that quorum‐sensing dispersion could be an effective therapeutic strategy. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:55–61, 2010     

Appraisal of Biofilm Formation in Diabetic Foot Infections by Comparing Phenotypic Methods With the Ultrastructural Analysis

Diabetic patients are more prone to the development of foot ulcers, because their underlying tissues are exposed to colonization by various pathogenic organisms. Hence, biofilm formation plays a vital role in disease progression by antibiotic resistance to the pathogen found in foot infections. The present study has demonstrated the correlation of biofilm assay with the clinical characteristics of diabetic foot infection. The clinical characteristics such as the ulcer duration, size, nature, and grade were associated with biofilm production. Our results suggest that as the size of the ulcer with poor glycemic control increased, the organism was more likely to be positive for biofilm formation. A high-degree of antibiotic resistance was exhibited by the biofilm-producing gram-positive isolates for erythromycin and gram-negative isolates for cefpodoxime. Comparisons of biofilm production using 3 different conventional methods were performed. The strong producers with the tube adherence method were able to produce biofilm using the cover slip assay method, and the weak producers in tube adherence method had difficulty in producing biofilm using the other 2 methods, indicating that the tube adherence method is the best method for assessing biofilm formation. The strong production of biofilm with the conventional method was further confirmed by scanning electron microscopy analysis, because bacteria attached as a distinct layer of biofilm. Thus, the high degree of antibiotic resistance was exhibited by biofilm producers compared with nonbiofilm producers. The tube adherence and cover slip assay were found to be the better method for biofilm evaluation.     

复配香辛料提取物体外阻断N-二甲基亚硝胺生成的效果

在确定桂皮、柚子皮、八角和花椒4种香辛料中活性物质最佳提取条件的基础上,利用所提取的活性物质在不同浓度下进行阻断N-二甲基亚硝胺生成的体外试验,并以不同比例进行复配,根据阻断率选出最佳复配比.结果显示:当桂皮、柚子皮、八角、花椒提取液四者达最佳复配比时,复配液具有较好的阻断效果.     

Biofilm formation increases treatment failure in Staphylococcus epidermidis device‐related osteomyelitis of the lower extremity in human patients

The ability to form biofilm on the surface of implanted devices is often considered the most critical virulence factor possessed by Staphylococcus epidermidis in its role as an opportunistic pathogen in orthopaedic device‐related infection (ODRI). Despite this recognition, there is a lack of clinical evidence linking outcome with biofilm forming ability for S. epidermidis ODRIs. We prospectively collected S. epidermidis isolates cultured from patients presenting with ODRI. Antibiotic resistance patterns and biofilm‐forming ability was assessed. Patient information was collected and treatment outcome measures were determined after a mean follow‐up period of 26 months. The primary outcome measure was cure at follow‐up. Univariate logistic regression models were used to determine the influence of biofilm formation and antibiotic resistance on treatment outcome. A total of 124 patients were included in the study, a majority of whom (n = 90) involved infections of the lower extremity. A clear trend emerged in the lower extremity cohort whereby cure rates decreased as the biofilm‐forming ability of the isolates increased (84% cure rate for infections caused by non‐biofilm formers, 76% cure rate for weak biofilm‐formers, and 60% cure rate for the most marked biofilm formers, p = 0.076). Antibiotic resistance did not influence treatment cure rate. Chronic immunosuppression was associated with a statistically significant decrease in cure rate (p = 0.044). Clinical significance: The trend of increasing biofilm‐forming ability resulting in lower cure rates for S. epidermidis ODRI indicates biofilm‐forming ability of infecting pathogens does influence treatment outcome of infections of the lower extremity. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1905–1913, 2016.