Disruption of Staphylococcus epidermidis biofilm formation using a targeted cationic peptide

This study reports the use of a targeted cationic peptide with the ability to disrupt Staphylococcus epidermidis biofilm formation. Complications due to nosocomial infections of implanted medical devices pose a significant health risk to patients, with Staphylococcus epidermidis often implicated in the case of blood-contacting biomaterials. S. epidermidis virulence relies mainly on its ability to form a biofilm, the main component of which is polysaccharide intercellular adhesin (PIA). We utilized the synthetic β6-20 peptide, known to specifically bind S. epidermidis, in order to deliver a cationic polylysine peptide (G(3)K(6)) to the bacterial surface and disrupt the charge-charge interactions needed for PIA retention and biofilm stability. The effects of the β6-20-G(3)K(6) peptide on biofilm formation were assessed using optical density, fluorescently labeled wheat germ agglutinin, nucleic acid stain (SYTO 9), and a metabolic assay (XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt). Biofilms formed in the presence of β6-20-G(3)K(6) peptide (100 μM) resulted in a 37.9% reduction in PIA content and a 17.5% reduction of adherent bacteria relative to biofilms grown in the absence of peptide. These studies demonstrate the targeting ability of the β6-20 peptide towards biomaterial-adherent S. epidermidis, and highlight the potential for disrupting the early stages of biofilm formation.     

生物材料植入感染与表皮葡萄球菌生物膜形成的相关基因调控

表皮葡萄球菌是寄居于人体皮肤和黏膜表面的共生菌群,现已发现其是引发临床生物材料相关感染的主要条件致病菌,在生物材料植入感染中占有重要地位.医用生物材料表面细菌生物膜的形成是其主要致病因素,细菌生物膜可有效抵御机体的防御反应和抗生素治疗,导致生物材料植入感染难以彻底治愈,使感染呈慢性、持续性和反复性特点,从而在临床上造成了极高的死亡率.就表皮葡萄球菌生物膜的形成、胞间黏附素基因(ica)操纵子和附属基因调节子(agr)基因对表皮葡萄球菌生物膜的调控及其在临床生物材料植入感染中的作用等方面作一综述.     

表皮葡萄球菌临床株生物被膜形成及其icaA基因的分析

目的 检测表皮葡萄球菌临床株生物被膜的形成能力,了解icaA基因及其表达与生物被膜形成的关系.方法 收集205株临床分离表皮葡萄球菌,刚果红平板试验检测其黏附性,半定量黏附试验检测其生物被膜的形成能力,扫描电镜观察生物被膜形态,PCR方法 扩增icaA基因片段,RT-PCR方法 分析icaA基因表达情况.结果 205株表皮葡萄球菌中刚果红平板试验阳性24株,半定量黏附试验阳性22株,28株枪测到icaA基因.半定量黏附试验阳性菌株的icaA基因表达水平呈现高于半定量黏附试验阴性菌株的趋势.结论 表皮葡萄球菌临床株具有一定的形成生物被膜的能力,icaA基因的存在及其正常表达是表皮葡萄球菌形成生物被膜的重要分子生物学基础,icaA基因表达尚有其他因索调控.     

Role of the luxS quorum-sensing system in biofilm formation and virulence of Staphylococcus epidermidis

Nosocomial infections caused by Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. Quorum-sensing regulation plays a major role in the biofilm development of many bacterial pathogens. Here, we describe luxS, a quorum-sensing system in staphylococci that has a significant impact on biofilm development and virulence. We constructed an isogenic DeltaluxS mutant strain of a biofilm-forming clinical isolate of S. epidermidis and demonstrated that luxS signaling is functional in S. epidermidis. The mutant strain showed increased biofilm formation in vitro and enhanced virulence in a rat model of biofilm-associated infection. Genetic complementation and addition of autoinducer 2-containing culture filtrate restored the wild-type phenotype, demonstrating that luxS repressed biofilm formation through a cell-cell signaling mechanism based on autoinducer 2 secretion. Enhanced production of the biofilm exopolysaccharide polysaccharide intercellular adhesin in the mutant strain is presumably the major cause of the observed phenotype. The agr quorum-sensing system has previously been shown to impact biofilm development and biofilm-associated infection in a way similar to that of luxS, although by regulation of different factors. Our study indicates a general scheme of quorum-sensing regulation of biofilm development in staphylococci, which contrasts with that observed in many other bacterial pathogens.     

Prevention of Staphylococcus epidermidis biofilm formation using a low-temperature processed silver-doped phenyltriethoxysilane sol-gel coating

Sol-gel coatings which elute bioactive silver ions are presented as a potential solution to the problem of biofilm formation on indwelling surfaces. There is evidence that high-temperature processing of such materials can lead to diffusion of silver away from the coating surface, reducing the amount of available silver. In this study, we report the biofilm inhibition of a Staphylococcus epidermidis biofilm using a low-temperature processed silver-doped phenyltriethoxysilane sol-gel coating. The incorporation of a silver salt into a sol-gel matrix resulted in an initial high release of silver in de-ionised water and physiological buffered saline (PBS), followed by a lower sustained release for at least 6 days-as determined by graphite furnace-atomic absorption spectroscopy (GF-AAS). The release of silver ions from the sol-gel coating reduced the adhesion and prevented formation of a S. epidermidis biofilm over a 10-day period. The presence of surface silver before and after 24 h immersion in PBS was confirmed by X-ray photoelectron spectroscopy (XPS). These silver-doped coatings also exhibited significant antibacterial activity against planktonic S. epidermidis. A simple test to visualise the antibacterial effect of silver release coatings on neighbouring bacterial cultures is also reported.     

Effect of cast molded rifampicin/silicone on Staphylococcus epidermidis biofilm formation

Infection is one of the most common catheter-related complications, especially in shunt systems used to treat hydrocephalus. Staphylococcus epidermidis is directly related to biomaterial infections owing to its ability to form a biofilm on implanted materials. In this study, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to investigate the effect of the antibiotic rifampicin on the colonization and growth of S. epidermidis 35984 on the surface of silicone. A cast molding method was used to load rifampicin into the silicone precursor before it was cured. Bacteria with a diameter of 800-1000 nm and height of 200-500 nm were found to be embedded in the biofilm. Compact multilayer biofilm structures were found on silicone surfaces upon incubation for 4 and 24 h. On the other hand, sparser biofilm structures were observed on rifampicin-loaded surfaces after incubation for the same duration. Deformation of bacteria was observed by AFM. Moreover, different bacterial colony structures on the surfaces of silicone and rifampicin-loaded silicone were observed by AFM and SEM.     

Ciprofloxacin-releasing bioabsorbable polymer is superior to titanium in preventing Staphylococcus epidermidis attachment and biofilm formation in vitro

Antibiotic coating systems have been successfully used to prevent bacterial attachment and biofilm formation. Our purpose was to evaluate whether bioabsorbable polylactide-co-glycolide (PLGA) 80/20 on its own, and PLGA together with ciprofloxacin (PLGA+C) have any advantages over titanium in preventing Staphylococcus epidermidis attachment and biofilm formation in vitro. Cylindrical specimens of titanium, PLGA, and PLGA+C in triplicate were examined for S. epidermidis ATCC 35989 attachment and biofilm formation after incubation with a bacterial suspension of about 10(5) cfu/mL for 1, 3, 7, 14, and 21 days, using scanning electron microscopy. Growth inhibition properties of PLGA and PLGA+C cylinders were tested on agar plates. On days 1, 3, and 21, no bacterial attachment was seen in 19.5, 9.2, and 41.4% of the titanium specimens; in 18.4, 28.7, and 34.5% of the PLGA specimens; and in 57.5, 62.1, and 57.5% of the PLGA+C specimens, respectively. During the whole study period, no biofilm was observed on 74-93% of the titanium specimens, 58-78% of the PLGA specimens, and 93-100% of the PLGA+C specimens. PLGA+C showed clear bacterial growth inhibition on agar plates, while PLGA and titanium did not show any inhibition. PLGA+C bioabsorbable material was superior to titanium in preventing bacterial attachment and biofilm formation and may have clinical applicability, for example, in prevention of infection in trauma surgery or in the treatment of chronic osteomyelitis.     

表皮葡萄球菌生物膜形成与ica操纵子的相关性研究

目的　研究表皮葡萄球菌 (表葡 )生物膜形成与ica操纵子存在之间的相关性并比较 3种检测表皮葡萄球菌生物膜形成的方法。方法　通过PCR法扩增icaA基因以检测ica操纵子的存在。用半定量粘附实验、扫描电子显微镜法和光学显微镜法检测自临床分离到的 19株表葡的粘附能力。结果　在 19株表葡中 ,通过PCR法测得 7株为icaA阳性 ,通过半定量粘附实验测得 5株icaA(+)菌株为粘附株 ;大多数菌株在含糖培养基中所测得的吸光度 (A)值大于不含糖培养基中所测得的A值。结论　ica操纵子的存在与表葡生物膜形成在统计学上显著相关 (P <0 .0 2 5 )。培养基中补充葡萄糖有利于大多数表葡的粘附。半定量粘附实验和光镜检测法适用于细菌粘附的初步测定和筛选 ,而扫描电镜检测法有更高的灵敏度以区分粘附菌株和非粘附菌株。     

Virulence gene expression by Staphylococcus epidermidis biofilm cells exposed to antibiotics

Staphylococcus epidermidis have become important causes of nosocomial infections, as its pathogenesis is correlated with the ability to form biofilms on polymeric surfaces. Production of poly-N-acetylglucosamine (PNAG) is crucial for S. epidermidis biofilm formation and is synthesized by the gene products of the icaADBC gene cluster. Production of PNAG/polysaccharide intercellular adhesin and biofilm formation are regulated by the alternative sigma factor, σ(B), and is influenced by a variety of environmental conditions including disinfectants and other antimicrobial substances. The susceptibility of five S. epidermidis strains to antibiotics alone and in double combination was previously tested. Our results demonstrated that some combinations are active and present a general broad spectrum against S. epidermidis biofilms, namely rifampicin-clindamycin and rifampicin-gentamicin. In the present study, it was investigated whether the combination of rifampicin with clindamycin and gentamicin and these antibiotics alone influence the expression of specific genes (icaA and rsbU) of S. epidermidis within biofilms using real-time polymerase chain reaction. The data showed that in most cases the expression of both genes tested significantly increased after exposure to antimicrobial agents alone and in combination. Besides having a similar antimicrobial effect, rifampicin combined with clindamycin and gentamicin induced a lower expression of biofilm-related genes relatively to rifampicin alone. Associated with the advantage of combinatorial therapy in avoiding the emergence of antibiotic resistance, this study demonstrated that it can also cause a lower genetic expression of icaA and rsbU genes, which are responsible for PNAG/polysaccharide intercellular adhesin production, and consequently reduce biofilm formation recidivism, relatively to rifampicin alone.     

Extracellular DNA-dependent biofilm formation by Staphylococcus epidermidis RP62A in response to subminimal inhibitory concentrations of antibiotics

We measured the ability of Staphylococcus epidermidis to form biofilms in the presence of subminimal inhibitory concentrations (sub-MICs) of vancomycin, tigecycline, linezolid and novobiocin. Six strains that produce different amounts of biofilm were tested. The three strains that produced the highest amounts of biofilm exhibited steady-state or decreased biofilm formation in the presence of sub-MIC antibiotics, whereas the three strains that produced lower amounts of biofilm exhibited up to 10-fold-increased biofilm formation in the presence of sub-MIC antibiotics. In two of the inducible strains (9142 and 456a), antibiotic-induced biofilm formation was inhibited by dispersin B, an enzyme that degrades poly-N-acetylglucosamine (PNAG) biofilm polysaccharide. In the third inducible strain (RP62A), dispersin B inhibited biofilm formation in response to sub-MIC vancomycin, but not to sub-MIC tigecycline. In contrast, DNase I efficiently inhibited biofilm formation by strain RP62A in response to sub-MIC tigecycline and vancomycin. DNase I had no effect on antibiotic-induced biofilm formation in strains 9142 and 456a. Our findings indicate that antibiotic-induced biofilm formation in S. epidermidis is both strain- and antibiotic-dependent and that S. epidermidis RP62A utilizes an extracellular DNA-dependent mechanism to form biofilms in response to sub-MIC antibiotics.     

Effects of biomaterial surface chemistry on the adhesion and biofilm formation of Staphylococcus epidermidis in vitro

The formation of biofilm, a structured community of bacteria enclosed in slime, is a significant virulence factor in medical-device-centered infection. The development of cardiovascular device infection can be separated into two phases: initial bacterial adhesion and aggregation, followed by proliferation and production of slime. It is possible to modulate the adhesion and biofilm formation of Staphylococcus epidermidis, a commensal skin bacterium commonly found on infected medical devices, through biomaterial surface chemistry. This study examines bacterial adhesion and biofilm formation on surface-modified polyethylene terephthalate (PET), including surfaces with varying hydrophilic, hydrophobic, and ionic character. Bacterial adhesion and biofilm formation were observed over 48 hours in phosphate-buffered saline (PBS) and 20% pooled human serum. The hydrophilic surface (PAAm) had significantly less nonspecific adhesion of bacteria than that in the control (PET) and other surfaces, when cultured in PBS (P < 0.0001). Charged surfaces, both anionic and cationic, had increased adhesion and aggregation of bacteria in comparison with the control (PET) in the presence of serum proteins over 24 hours (P < 0.0001). Bacteria cultured in serum on the charged surfaces did not have significantly different amounts of biofilm formation compared with that of the control (PET) surface after 48 hours. This study showed that biomaterial surface chemistry characteristics impact initial adhesion and aggregation of S. epidermidis on biomaterials.     

RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3

The σ^S subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses.     

cid和lrg基因表达与表皮葡萄球菌临床株生物被膜形成关系的研究

目的 cid和lrg操纵子是细菌程序性死亡和溶解的重要调控基因,本研究旨在探究表皮葡萄球菌临床株cidA和lrgA基因及其mRNA表达水平与生物被膜形成的关系.方法 前期收集的表皮葡萄球菌(来源于血液、尿液、痰、分泌物及导管等生物材料表面)临床株39株,通过黏附试验和icaA基因扩增将其分为生物被膜形成菌株20株,非生物被膜形成菌株19株,PCR扩增表皮葡萄球菌cidA和lrgA基因片段,半定量RT-PCR检测cidA和lrgA mRNA水平,计算cid/lrg mRNA比率.结果 39株表皮葡萄球菌临床株均可扩增出cidA和lrgA的基因片段.随机选取生物被膜形成菌株表皮葡萄球菌Y36和非生物被膜形成菌株表皮葡萄球菌Y26检测培养2、4、6、8、10 h的cidA和lrgA的表达水平,两株菌cidA mRNA均在培养4 h后达峰,lrgA mRNA均在培养6 h后达峰.39株表皮葡萄球菌培养4 h后,生物被膜形成菌株与非生物被膜形成菌株,菌株中cidA基因相对表达水平分别为0.340±0.250和0.406±0.408(P=0.541),lrgA基因相对表达水平为0.325±0.218和0.253±0.211(P=0.299),两组相比差异均无统计学意义.cid/lrg mRNA比率,生物被膜形成菌株为1.067±0.529,非生物被膜形成菌株为1.958±1.877,两组总体分布差异有统计学意义(P=0.001).结论 表皮葡萄球菌临床株cidA和lrgA基因表达可能具有一定的时限性,cidA和lrgA基因在生物被膜形成菌株与非生物被膜形成菌株表皮葡萄球菌的表达没有明显差异,cid/lrg mRNA比率可能具有一定生物学意义.     

Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.     

The control of Staphylococcus epidermidis biofilm formation and in vivo infection rates by covalently bound furanones

In order to overcome the continuing infection rate associated with biomaterials, the use of covalently bound furanones as an antibiofilm coating for biomaterials has been investigated. Furanones have previously been shown to inhibit growth of Gram-positive and Gram-negative bacteria. The aim of these studies were to covalently bind furanones to polymers and to test their efficacy for inhibiting biofilm formation of Staphylococcus epidermidis and in vivo infection rate. Two methods of covalent attachment of furanones were used. The first, a co-polymerisation with a styrene polymer, and second, a plasma-1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) reaction to produce furanone-coated catheters. Biofilm formation by S. epidermidis in vitro was inhibited by 89% for polystryene-furanone disks and by 78% by furanone-coated catheters (p<0.01). In an in vivo sheep model we found furanones were effective at controlling infection for up to 65 days. Furanones have potential to be used as a coating for biomaterials to control infection caused by S. epidermidis.     

Inhibition of biofilm formation by monoclonal antibodies against Staphylococcus epidermidis RP62A accumulation-associated protein

Staphylococcus epidermidis expresses a 140-kDa cell wall-bound protein accumulation-associated protein (AAP) to adhere to and accumulate as a biofilm on a surface. Potentially blocking AAP with a monoclonal antibody (MAb) could reduce or eliminate S. epidermidis bacterial colonization of biomedical devices. Here, we report on our efforts to (i) isolate AAP, (ii) generate MAbs against AAP, and (iii) determine the efficacy of MAbs to inhibit S. epidermidis biofilm formation. An M7 S. epidermidis mutant, reportedly deficient in AAP expression, was used as a negative control. Postinoculation murine sera, containing polyclonal antibodies against AAP, were able to reduce S. epidermidis biofilm formation by 54%. Select MAbs against AAP were able to reduce S. epidermidis by no more than 66%. Two MAb mixtures, 12C6/12A1 and 3C1/12A1, reduced S. epidermidis accumulation up to 79 and 87%, respectively, significantly more than individual MAbs. Contrary to a previous report, biofilm-deficient S. epidermidis mutant M7 expressed a 200-kDa protein on its cell wall that specifically bound AAP MAbs. Peptide characterization of this M7 protein by microcapillary reversed-phase high-pressure liquid chromatography-nanoelectrospray tandem mass spectrometry resulted in 53% homology with AAP. Ongoing studies will elucidate the dynamic expression of AAP and the M7 200-kDa protein in order to define their roles in biofilm formation.     

表皮葡萄球菌生物膜形成与生物材料感染

随着各种生物材料的应用，慢性感染及医源性感染日益突出，凝固酶阴性球菌-表皮葡萄球菌成为首要病原体，它主要致病机制是生物膜形成，生物膜形成则成为生物材料感染难以控制的根源。细菌生物膜是具有高度组织化的多细胞群体结构，它们间相互通讯，有着精密的调控机制以适应不同的环境，能有效抵御机体的防御反应和抗生素治疗。随着对生物膜分子水平研究的不断深入，为临床防治生物膜相关性感染提供了更有效的靶点。