Influence of batch or fed-batch growth on Staphylococcus epidermidis biofilm formation

AIMS: To make a quantitative evaluation of the differences in biofilm formation by Staphylococcus epidermidis using batch and fed-batch growth systems and to correlate this with production of the major biofilm polysaccharide, poly-N-acetyl glucosamine (PNAG). METHODS AND RESULTS: Dry weight measurements of biofilms formed in batch and fed-batch conditions were compared with haemagglutination titres, which measure the amount of PNAG produced. Strains grown in batch systems developed less biofilm than when grown in fed-batch systems. A good correlation was found between the amount of biofilm formed in fed-batch systems and the haemagglutination titres. CONCLUSIONS: Differences in biofilm formation and PNAG production by S. epidermidis are dependent on the availability of nutrients, with higher availability correlating with more biofilm and PNAG production. SIGNIFICANCE OF AND IMPACT OF THE STUDY: Comparisons of the formation of biofilms by S. epidermidis are dependent on choosing an appropriate biofilm growth system. Comparability or disparity of conclusions among different investigations will be strongly influenced by which mode S. epidermidis biofilms are formed.     

表皮葡萄球菌 YycG单克隆抗体抗生物膜活性的研究

YycFG双组分信号转导系统调控葡萄球菌细胞壁合成、代谢及生物膜形成,PAS是组氨酸激酶YycG的胞外信号感应区域。本研究针对胞外PAS区域制备抗YycG‐PAS单克隆抗体（YycG‐PAS MAb ）。免疫鉴定结果表明,YycG‐PAS MAb可与重组YycG‐PAS蛋白和表皮葡萄球菌菌体 YycG蛋白结合,属于IgG2a亚型,效价为log 215。YycG‐PAS MAb（80μg／ml）与表皮葡萄球菌 RP62A菌株共培养24 h ,对细菌生物膜形成的抑制率达46．5％,与 mIgG （80μg／mL ）组相比有显著差异（ P＜0．05）。YycG‐PAS MAb （80μg／ml）与低浓度万古霉素（1μg／ml）联用可增强对24 h细菌生物膜的抑制作用,抑制率由57．6％提高至93．0％（P＜0．01）。YycG‐PAS MAb对浮游状态表皮葡萄球菌的生长及存活无显著影响。本研究为YycG‐PAS MAb在抗生物膜感染中的潜在应用价值奠定了一定基础。     

连翘苷和黄芩苷对表皮葡萄球菌生物膜抑制作用的研究

目的通过中药有效成分连翘苷和黄芩苷分别对表皮葡萄球菌生物膜抑制作用的研究,为表皮葡萄球菌生物膜引起的相关感染提供新的治疗途径。方法体外构建表皮葡萄球菌生物膜,XTT减低法评价连翘苷、黄芩苷对表皮葡萄球菌初始黏附及生物膜内细菌代谢的影响,显微镜下观察用药后表皮葡萄球菌生物膜形态和结构改变。结果连翘苷和黄芩苷对表皮葡萄球菌生物膜的早期黏附均无抑制作用;连翘苷对表皮葡萄球菌生物膜菌的SMIC50为31.25μg/ml,而黄芩苷对表皮葡萄球菌生物膜菌的代谢无影响;在显微镜下观察,连翘苷使部分表皮葡萄球菌被膜的形态发生改变,而黄芩苷对其形态影响不显著。结论连翘苷对表皮葡萄球菌生物膜的初始黏附阶段无抑制作用,对生物膜菌的代谢和生物膜形态均有显著影响;黄芩苷对表皮葡萄球菌生物膜无显著作用。     

In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.     

2-Hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus is antagonistic to Staphylococcus epidermidis biofilm formation

Abstract

Staphylococcus epidermidis (SE) is an opportunistic nosocomial pathogen that accounts for recalcitrant device-related infections worldwide. Owing to the growing interest in plants and their secondary metabolites targeting bacterial adhesion, this study was intended to uncover the anti-biofilm potential of Hemidesmus indicus and its major constituent 2-hydroxy-4-methoxybenzaldehyde (HMB) against SE. The minimum biofilm inhibitory concentration (MBIC) of H. indicus root extract and HMB were found to be 500 and 250?µg ml^?1, respectively. The results of time-dependent biofilm inhibition and mature biofilm disruption assays confirmed that HMB targets initial cell adhesion. Furthermore, interference by HMB in the expression of adhesin genes (icaA, aap and bhp) and biofilm components was associated with an increased susceptibility of SE to oxidative stress and antibiotics. To conclude, this study reports for the first time HMB as a potential drug against SE biofilms.     

纳米银离子对表皮葡萄球菌生物膜形成过程的影响

目的探讨纳米银离子对表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm)形成过程的影响。方法采用摇床法,以纳米银离子含量不同的乙烯-醋酸乙烯酯(Ethylene-Vinyl acetate,EVA)塑料为细菌粘附载体,建立体外生物膜模型;将各个时间点培养好的标本放在扫描电子显微镜(Scanning Electron Microscopy,SEM)及倒置显微镜下观察空白组与纳米银离子干预组中生物膜的形成情况,并结合NIS-Element BR软件计算生物膜覆盖率(biofilm coverage rate,BCR);荧光探针SYTO9/PI标记生物膜内细菌、激光共聚焦显微镜(Confocal LaserScanning Microscopy,CLSM)结合生物膜图形结构分析软件(Image Structure Analyze,ISA)观察纳米银离子作用后各时间点生物膜的结构变化。结果 2 d组生物膜经SEM检测,纳米银离子干预组较空白对照组相比,细菌变稀疏,结构明显受到破坏。BCR检测,0.5 d时纳米银离子干预组较空白对照组相比差异没有统计学意义,但在1 d、2 d时BCR差异有统计学意义(P<0.05)。CLSM结合ISA软件分析结果显示,2 d时空白对照组与0.1%纳米银离子干预组生物膜厚度、平均扩散距离(average diffusion distance,ADD)和结构熵(textual entropy,TE),分别为17.55±2.08和11.33±0.98、3.12±0.30和1.93±0.13、5.79±和3.17±0.16(P<0.05);区域孔率(Areal porosity,AP)为0.90±0.01和0.98±0.01(P<0.05)。但5 d时2组参数之间差异没有统计学意义。0.05%纳米银离子组也有相同的趋势。结论纳米银离子对表皮葡萄球菌的形成有抑制作用,但随着时间的推移,其抗菌作用逐渐减弱。高浓度组较低浓度组抗菌效果更加明显,持续时间更长。     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型

目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。     

Lack of biofilm contribution to bacterial colonisation in an experimental model of foreign body infection by Staphylococcus aureus and Staphylococcus epidermidis

The contribution of in vivo biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis was studied in an experimental model of foreign body infections. Increasing inocula (from 10(2) to 10(7) organisms) of ica-positive strains of S. aureus and S. epidermidis and their ica-negative isogenic mutants (the ica locus codes for a major polysaccharide component of biofilm) were injected into subcutaneously implanted tissue cages in guinea pigs. Surprisingly, bacterial counts and time-course of tissue cage infection by ica-positive strains of S. aureus or S. epidermidis were equivalent to those of their respective ica-negative mutants, in the locally infected fluids and on tissue-cage-inserted plastic coverslips.     

Cloning and expression of Staphylococcus epidermidis urease gene sequences in Staphylococcus carnosus

The urease gene sequences of Staphylococcus epidermidis CNS23 were cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned sequences revealed four polypeptides (60, 17, 11 and 7.5 kDa) that were associated with enzyme activity. Southern hybridisation experiments showed high homologies with the urease genes of Staphylococcus saprophyticus.     

Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as ‘negative for biofilm formation’ based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.     

亚抑菌浓度的穿心莲内酯及与红霉素联用影响表皮葡萄球菌生物膜形成的初步研究

目的 观察亚抑菌浓度(亚-MIC)穿心莲内酯及其与红霉素联用对表皮葡萄球菌生物膜形成的影响.方法 连续稀释法测定穿心莲内酯和红霉素对表皮葡萄球菌的MIC;棋盘格法测定穿心莲内酯和红霉素联用对表皮葡萄球菌悬浮菌的作用;体外构建表皮葡萄球菌生物膜,XTT减低法评价亚-MIC穿心莲内酯及与红霉素联用对表皮葡萄球菌生物膜内细菌代谢及初始黏附能力的影响,扫描电镜观察用药后表皮葡萄球菌形态和生物膜结构改变.结果 穿心莲内酯对表皮葡萄球菌悬浮菌的MIC大于1000 mg/L,穿心莲内酯和红霉素联用对表皮葡萄球菌悬浮菌的FIC指数(联合抑菌分数)＞2;亚抑菌浓度穿心莲内酯对表皮葡萄球菌生物膜黏附及膜内菌代谢均有抑制作用;穿心莲内酯和红霉素联用对表皮葡萄球菌的黏附能力的影响表现为协同作用,对悬浮菌和膜内菌代谢作用表现为拮抗.结论 亚抑菌浓度穿心莲内酯对表皮葡萄球菌生物膜黏附及膜内菌代谢均有抑制作用;穿心莲内酯和红霉素联用对表皮葡萄球菌悬浮菌及膜内菌代谢存在拮抗,提示临床治疗表皮葡萄球菌感染时应避免同时使用这两种药物.     

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL^-1, including 15.7% with an MIC of 8 μg mL^-1 and 2% with an MIC of 16 μg mL^-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.     

Characterization of a modified rotating disk reactor for the cultivation of Staphylococcus epidermidis biofilm

Aims: The purpose of this study was to develop a system that would allow biofilms to be cultivated under strictly defined conditions in terms of dissolved oxygen, fluid shear and to assess whether the method was suitable for the detection of respiratory activity stratification in biofilm samples. Methods: The system is a modified version a commercially available laboratory biofilm reactor and incorporates a number of features such as the provision of defined levels of dissolved oxygen, constant average shear, enhanced gas–liquid mass transfer, aseptic operation and the ability to remove biofilm for ex situ analysis during or after continuous cultivation. Conclusions: The system was shown to be effective for the characterization of the effects of dissolved oxygen on a pure culture of Staphylococcus epidermidis. The versatility of the system offers the potential for cultivating pure culture biofilm in defined, controlled conditions and facilitates a range of analyses that can be performed ex situ. Significance and Impact of the Study: The ability to provide strict regulation of environmental conditions and enhanced transfer of oxygen to the biofilm during cultivation are important, first because oxygen is known to regulate biofilm development in several micro‐organisms and second because many conventional biofilm cultivation systems may not provide adequate oxygen supply to the biofilm.     

穿心莲内酯等中药有效成分对表皮葡萄球菌生物膜渗透性的比较

目的比较穿心莲内酯、大黄素、盐酸小檗碱等对表皮葡萄球菌生物膜的渗透性,为中药治疗表皮葡萄球菌引起的相关感染提供相关研究的依据。方法在体外建立SE—BF（表皮葡萄球菌生物膜模型）和PB（生物膜渗透模型）,观察穿心莲内酯、大黄素、盐酸小檗碱等中药有效成分对PB的渗透率。结果大黄素、穿心莲内酯36h渗透率分别达到88．00％、82．89％,而盐酸小檗碱渗透率在几种药物中相对较差。结论中药有效成分对表皮葡萄球菌生物膜有较强的渗透性。     

眼分泌物耐甲氧西林表皮葡萄球菌检测及耐药性分析

目的 探讨眼科分泌物中表皮葡萄球菌耐药特征,指导临床合理使用抗菌药物.方法 用定量接种针挑取单个菌落溶于0.5 McFarland硫酸钡接种液制成混悬液,再用接种器吸取菌液接种于Pos Combo Panel Type20,35℃孵育24 h,应用美国德灵SCAN4微生物分析仪对192株葡萄球菌中的86株表皮葡萄球菌进行鉴定和药敏试验.结果 表皮葡萄球菌检出86株,检出率为44.8％ (86/192);MRSE检出58株,检出率为30.2％ (58/192),MRSE占表皮葡萄球菌的67.4％ (58/86);MRSE对头孢西丁、青霉素、红霉素、阿莫西林/克拉维酸、头孢唑啉、复方新诺明、苯唑西林、左氧氟沙星、庆大霉素、亚胺培南具有显著耐药性,对万古霉素、利奈唑烷、利福平、四环素、氯霉素敏感.结论 眼科分泌物中表皮葡萄球菌耐药性严重,并且MRSE呈现多重耐药,医院应加强对MRSE的检测,依据药敏结果合理选用抗菌药物治疗感染.     

Rapid depletion of dissolved oxygen in 96‐well microtiter plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration

Biofilm‐related research using 96‐well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96‐well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96‐well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96‐well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model. Biotechnol. Bioeng. 2009;103: 1042–1047. © 2009 Wiley Periodicals, Inc.     

Surface free energy and interaction of Staphylococcus epidermidis with biomaterials

The adhesion of twenty nine Staphylococcus epidermidis strains to teflon, polyethylene, polycarbonate and bovine pericardium was studied in vitro and examined in relation to the surface free energies of both bacteria and biomaterials. All S. epidermidis strains had similar surface free energies, close to that of water, and adhered better to the materials with analogous surface free energies. There was a significant correlation (Kendall's Tau B = 1000) of biomaterial's surface free energy with the number of adhering bacteria. This correlation is inverse (Kendall's Tau B = -1000) when surface hydrophobicity is considered instead of surface free energy. This indicates that in Staphylococcus epidermidis adherence to biomaterials is inversely correlated to the surface hydrophobicity of the last, being so just the opposite of that occurring with other bacteria.     

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