香芹酚抑制金黄色葡萄球菌生物被膜的形成

【背景】生物被膜是细菌的一种自我保护形式,可以增强细菌对药物及宿主免疫应答的抵抗力,引起细菌耐药性和持续性感染。【目的】探究香芹酚对金黄色葡萄球菌生物被膜的作用机制,为开发新型抗生物被膜药物提供可靠的理论依据。【方法】通过结晶紫染色法检测香芹酚对供试菌株生物被膜形成的抑制和对成熟生物被膜的清除作用;使用刚果红平板法探究香芹酚对供试菌株生物被膜形成过程中细胞间多糖黏附素(polysaccharide intercellular adhesion,PIA)合成的作用;通过分光光度法检测香芹酚对胞外DNA (extracellular DNA,eDNA)分泌的抑制作用;利用RT-PCR技术检测香芹酚对供试菌株的生物被膜相关基因icaA、cidA和sarA转录水平的影响。【结果】香芹酚对生物被膜形成的抑制和生物被膜的清除均有较强作用效果。256μg/mL香芹酚抑制PIA合成和e DNA释放的效果显著。香芹酚可通过抑制相关基因转录从而抑制生物被膜的形成,当64μg/mL的香芹酚作用后,sarA的转录水平降低了60.44%±2.91%,cidA的转录水平降低了76.48%±1.67%,icaA的转...     

苦参碱对表皮葡萄球菌生物被膜作用初探

通过中药有效成分苦参碱对表皮葡萄球菌生物被膜抑制作用的研究,为表皮葡萄球菌生物被膜引起的相关感染提供新的治疗途径。利用XTT减低法评价苦参碱对表皮葡萄球菌初始粘附及生物被膜内细菌代谢的影响,镜下观察该药对表皮葡萄球菌生物被膜的形态学影响。结果表明:苦参碱对表皮葡萄球菌生物被膜菌的SMIC50和SMIC80分别为62.5 mg/L和500 mg/L;1 000 mg/L浓度的苦参碱对表皮葡萄球菌早期粘附有抑制作用;250 mg/L浓度的苦参碱对表皮葡萄球菌生物被膜的形态有显著影响。因此可见,苦参碱对表皮葡萄球菌生物被膜的形成与粘附均有抑制作用。     

十二烷基苯磺酸钠对产膜表皮葡萄球菌黏附作用影响初探

目的通过阴离子表面活性剂十二烷基苯磺酸钠针对表皮葡萄球菌生物被膜菌黏附抑制作用的研究,为临床抗表皮葡萄球菌生物被膜菌引起的相关感染探索新的研究方向和可能的治疗途径。方法以红霉素为阳性对照,利用XTT减低法评价十二烷基苯磺酸钠对表皮葡萄球菌初始黏附的影响。结果十二烷基苯磺酸钠在1 000、100mg/L浓度下对表皮葡萄球菌初始黏附均有抑制作用;以浓度为1 000mg/L十二烷基苯磺酸钠对实验材料进行预处理,对表皮葡萄球菌初始黏附有明显抑制作用。结论阴离子表面活性剂十二烷基苯磺酸钠在特定浓度下对表皮葡萄球菌产生物被膜菌的初始黏附有抑制作用。     

表皮葡萄球菌vraSR-srrAB突变株的构建及生物学表型

【背景】形成生物被膜是表皮葡萄球菌的主要致病方式,双组分系统(two-component regulatory system, TCS) VraSR和SrrAB与细菌的生长、生物被膜形成等多种生物学表型密切相关。敲除表皮葡萄球菌SE1457 vraSR后细胞壁变薄,生物被膜形成量降低,而敲除srrAB后生长进入稳定期的时间延长,生物被膜形成量也降低,且TCS-VraSR和SrrAB均通过ica途径调控表皮葡萄球菌生物被膜的形成。ica操作子是表皮葡萄球菌生物被膜形成的重要调节元件,由icaADBC这4个开放阅读框(openreadingframe,ORF)和1个转录方向相反的icaR组成,IcaR是icaADBC的抑制子。【目的】探索TCS-VraSR和SrrAB协同调控表皮葡萄球菌生长及生物被膜形成中的作用,为防控表皮葡萄球菌引起的持续性感染奠定基础。【方法】构建pKOR1-ΔvraSR重组质粒,经大肠杆菌DC10B修饰后转入ΔsrrAB,通过同源重组在ΔsrrAB突变株的基础上进一步敲除vraSR基因,疑似ΔvraSR-srrAB突变株经PCR、RT-PCR和测序验证。检测Δvra...     

抗菌肽17BIPHE2对金黄色葡萄球菌生物被膜的抑制作用

【目的】研究抗菌肽17BIPHE2单独使用及联合抗生素对金黄色葡萄球菌(Staphylococcus aureus)生物被膜的抑制作用。【方法】采用刚果红平板测试法和结晶紫染色评估受试菌形成生物被膜的能力;微量肉汤稀释法和琼脂平板测试法测定金黄色葡萄球菌最小抑菌浓度(MIC)和最小杀菌浓度(MBC);利用抑制金黄色葡萄球菌黏附实验和生物被膜形成抑制实验观察17BIPHE2单独使用及联合抗生素对生物被膜黏附阶段和形成阶段的影响;通过扫描电子显微镜(SEM)观察17BIPHE2单独使用及联合抗生素对成熟生物被膜的清除作用。【结果】17BIPHE2的MIC为8μmol/L,1/2×MIC就可以有效抑制浮游菌的生长。单独使用17BIPHE2在细菌黏附阶段抑制率为40%,在生物被膜形成阶段抑制率达到35%。17BIPHE2联合抗生素使用较单独使用抗生素其抑制率均有所下降。生物被膜成熟阶段17BIPHE2于1/4×MIC浓度即可促进生物被膜崩解,1×MIC生物被膜崩解同时细菌黏附量有所下降,联合万古霉素促进生物被膜崩解同时细菌胞质大量外泄。【结论】抗菌肽17BIPHE2具有良好的抑制金黄色葡萄球菌生物被膜作用,联合抗生素其抗生物被膜作用进一步提高。这将为治疗由金黄色葡萄球菌生物被膜引起的相关感染提供了一个新思路。     

表面活性剂溴化十六烷基吡啶对产膜表皮葡萄球菌粘附作用的影响

目的 通过表面活性剂溴化十六烷基吡啶抗表皮葡萄球菌生物被膜菌粘附作用的研究,为临床针对产膜表皮葡萄球菌引起的相关感染提供可能的探索方向和防治途径。方法 以万古霉素为阳性对照,利用XTT减低法,评价溴化十六烷基吡啶对表皮葡萄球菌初始粘附的影响。结果 浓度为4、2和1 mg/L的溴化十六烷基吡啶对表皮葡萄球菌初始粘附均有显著的抑制作用,与阴性对照组比较差异有统计学意义(t=0.4555~34.9664,P<0.01);用浓度为32、16、8和4 mg/L的溴化十六烷基吡啶对实验材料预处理后,对表皮葡萄球菌初始粘附抑制作用明显,与阴性对照组比较差异有统计学意义(t=1.1125~21.9246,P<0.001)。结论 溴化十六烷基吡啶无论是直接作用或是对实验材料预处理,对表皮葡萄球菌形成生物被膜的初始粘附过程具有显著抑制作用。     

苦参碱、鱼腥草素钠及其与红霉素联用对表皮葡萄球菌生物被膜渗透作用的研究

目的比较苦参碱、鱼腥草素钠及其与红霉素联用对表皮葡萄球菌生物被膜的渗透性,为中药及中西药联用治疗表皮葡萄球菌引起的相关感染提供依据。方法在体外建立SE-BF(表皮葡萄球菌生物被膜模型),PB(生物被膜渗透模型);观察并测量含药滤纸片抑菌圈直径(每个时间点取样3个),计算苦参碱、鱼腥草素钠及其与红霉素联用对PB的渗透率。结果苦参碱、鱼腥草素钠、苦参碱与红霉素及鱼腥草素钠与红霉素联用对Se738的36h渗透率分别达到93.94%、96.97%、89.34%和93.53%;苦参碱、鱼腥草素钠、苦参碱与红霉素及鱼腥草素钠与红霉素联用对ATCC 35984的36h渗透率分别达到82.83%、88.55%、91.47%和96.76%。结论苦参碱、鱼腥草素钠及其与红霉素联用,对表皮葡萄球菌生物被膜均有较强的渗透性。     

葡萄糖对表皮葡萄球菌生物被膜形成的影响及调节机制的研究

生物被膜(Biofilm)是条件致病菌表皮葡萄球菌(Staphylococcusepidermidis)的主要致病因素,生物被膜的形成依赖多糖PIA合成,合成PIA的糖基转移酶由icaADBC基因编码。以生物被膜形成能力不同的菌株为对象,通过研究不同环境对生物被膜形成、细菌总糖量及相关基因表达的变化,探索外界环境对生物被膜形成的影响及葡萄糖对生物被膜诱导的分子机制。有利于生物被膜形成培养条件促进生物被膜形成及多糖的表达,葡萄糖能诱导ica基因的表达和生物被膜形成,ica基因的反义寡核苷酸(ODN)能对抗葡萄糖的作用;葡萄糖作用下不同生长周期生物被膜形成相关基因ica、icaR、AtlE表达不同。表皮葡萄球菌生物被膜的形成与细菌糖代谢有关,葡萄糖通过上调ica表达诱导生物膜形成,但不需要ica基因的持续表达;葡萄糖的诱导作用不是直接通过调节AtlE和icaR基因来实现的     

百里香酚联合苯唑西林对耐甲氧西林金黄色葡萄球菌(MRSA)生物被膜的影响

【背景】耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)能以生物被膜的状态存在,从而产生多重耐药性和持续性感染。【目的】通过研究百里香酚和苯唑西林单用和联用对耐甲氧西林金黄色葡萄球菌生物被膜的形成抑制和清除作用,探究联合用药对MRSA生物被膜的影响,为临床联合应用抗MRSA药物提供理论依据。【方法】采用微量肉汤稀释法测定苯唑西林对MRSA标准菌株USA300的最低抑菌浓度;采用结晶紫染色法和菌落计数法评估百里香酚和苯唑西林单用和联用对USA300生物被膜形成抑制和清除作用。【结果】百里香酚和苯唑西林在亚抑菌浓度下对USA300生物被膜的形成具有一定的抑制作用。在较高浓度下,百里香酚对其24 h和72 h形成的生物被膜有良好的清除作用,而苯唑西林无清除作用。两药联用对生物被膜的抑制和清除作用进一步增强,在较低浓度下有较好的抑制和清除效果。【结论】百里香酚和苯唑西林联合用药与单独用药相比,对USA300的生物被膜的抑制和清除作用增强,两药联合有协同抗菌作用。     

黄芩素抑制金黄色葡萄球菌生物被膜的形成

细菌生物被膜的形成是导致细菌耐药和引起持续性感染的主要原因之一。本文通过检测黄芩素对金黄色葡萄球菌26112菌株（Staphylococcus aureus 26112,SA26112）多糖细胞间黏附素（polysaccharide intercellular adhesion, PIA）的合成和胞外DNA（extracellular DNA,eDNA）释放量的影响,及其对icaA和cidA基因表达量的影响,探讨黄芩素对金黄色葡萄菌生物被膜形成的抑制作用及其机制。结果显示,黄芩素能抑制SA26112生物被膜的形成,其抑杀SA26112的最低抑菌浓度和最低杀菌浓度均为0.04 mg/mL。0.16 mg/mL黄芩素和256 μg/mL环丙沙星单独作用时,均不能杀死其成熟生物被膜内的SA26112细菌,而当二者联用时则可杀死成熟生物被膜内的细菌。黄芩素能显著抑制SA26112菌株PIA的合成、eDNA的释放量及icaA和cidA基因的相对表达量。其中,0.04 mg/mL黄芩素作用SA26112菌株24 h,与对照组相比,eDNA的释放量减少97%,icaA和cidA基因的相对表达量分别减少62%和41%。上述结果表明,黄芩素能抑制SA26112菌株生物被膜的形成,其作用机制可通过降低icaA和cidA的基因表达量,进而影响PIA的合成和eDNA的释放,来抑制金黄色葡萄球菌生物被膜的形成。     

Comparative proteomic analysis between the invasive and commensal strains of Staphylococcus epidermidis

Staphylococcus epidermidis is one of the major causative agents for nosocomial infections. To reveal the pathogenesis factors, we performed the comparative proteomic analysis of invasive ATCC35984 and commensal ATCC12228 strains by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. The differentially expressed proteins were involved in carbohydrate metabolism, sugar binding, lipid degradation and amino acid binding. In addition, we demonstrated that the trap gene was transcribed by 3.657+/-0.156 (P<0.01) -fold higher in ATCC35984 than in ATCC12228. Levels of accumulation-associated protein (AAP) were found to be low by the immuno-dot blotting assay in ATCC12228, which is unable to form biofilm. Our results suggest that the target of RNAIII activating protein and AAP may contribute to S. epidermidis virulence and biofilm formation.     

五倍子水煎剂对表皮葡萄球菌生物膜抑制的研究

通过五倍子水煎剂对表皮葡萄球菌MIC测定和生物膜形成干预的研究,为表皮葡萄球菌引起感染提供新的治疗途径。用微量肉汤稀释法分别测定五倍子水煎剂对表皮葡萄球菌的MIC;刚果红及刚果红红霉素、五倍子水煎剂琼脂平板测定表皮葡萄球菌PIA生成与抑制;五倍子水煎剂、红霉素干预表皮葡萄球菌生物膜形成,于光镜和电镜下观察其生物膜形态。134株表皮葡萄球菌五倍子水煎剂的MIC50为0.488 mg/mL,MIC90为0.977 mg/mL。134株表皮葡萄球菌中有50株为PIA阳性,PIA阳性的50株菌全部产生生物膜,红霉素对表皮葡萄球菌生物膜形成有抑制,而五倍子水煎剂则无。表皮葡萄球菌PIA的相互作用在其生物膜的生成中起主要作用;五倍子水煎剂对表皮葡萄球菌生长有明显的抑制但对生物膜形成无干预作用。     

Effect of allicin on the production of polysaccharide intercellular adhesin in Staphylococcus epidermidis

Aims: Polysaccharide intercellular adhesin (PIA) is the main agglutination agent in the biofilm forming strain Staphylococcus epidermidis. To find an explanation for the observed inhibition of biofilm formation by allicin, we studied the effect of allicin on PIA production in samples treated with sub MIC doses of allicin and compared this with a control culture without allicin. Methods and Results: Bacteria (Staph. epidermidis ATCC 35984) were grown in glass tubes, and PIA was extracted by vortex vibration using microbeads and NN dimethyl acetamide/LiCl as solvent. The extracts were filtered and passed through size exclusion columns. Chromatographic fractions were analysed with an excess of sodium metaperiodate and the excess was determined spectrophotometrically using 2,4,6‐tripyridyl‐s‐triazine. Conclusion: The amount of exopolysaccharides in samples previously treated with allicin is significantly lower than in the control. This finding suggests a specific enzymatic inhibition in PIA synthesis. Significance and Impact of the Study: This study provides an insight into the mechanism of biofilm formation, and is a biochemical model for PIA inhibition by allicin. The analysis proposed may be useful in studies of production of exopolysaccharides responsible for adherence and agglutination of Staph. epidermidis. Prevention of biofilm formation by allicin opens up a new field of in vitro studies and permits us to envisage future clinical applications.     

A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.     

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis

Aims: The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device‐related infections. Methods and Results: We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l^?1) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l^?1 concentration of LL37, indicating a direct effect of the peptide on biofilm production. Conclusions: As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. Significance and Impact of the Study: Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.     

Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.     

Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device-related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA-negative strains are not uncommon, suggesting the existence of PIA-independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation-associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap-negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide-independent manner. In contrast, expression of full-length Aap did not lead to a biofilm-positive phenotype. Obviously, to gain adhesive function, full-length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by alpha(2)-macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein-dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.