Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

表皮葡萄球菌生物被膜对红霉素渗透性的研究

为探讨表皮葡萄球菌生物被膜对红霉素的渗透性,我们采用生物被膜抗生素渗透模型检测Staphylcoccus epidermidis 1457、1457-msrA和临床分离株S68生物被膜不同时间点红霉素的渗透率,并用吖啶橙染色激光共聚焦显微镜观察生物被膜内细菌RNA/DNA的相对含量;扫描电子显微镜观察膜内细菌的密度.红霉素作用36 h后,1457、1457-msrA、S68的渗透率分别为0.93,0.55和0.4;1457渗透地较快,8 h后渗透率即达到0.58,而1457msrA和$68相对较为缓慢,24 h后分别为0.499和0.31:吖啶橙染色可见红霉素作用下膜内菌RNA和DNA的相对比例减小,生长速率下降;扫描电子显微镜观察可见生物被膜红霉素作用后空气面的细菌数与琼脂面相比均较少,细胞碎片相对较多,而对照组(无抗生素作用)琼脂面和空气面的细菌密度和分布较均匀.可见红霉素可渗透入表葡茵生物被膜,但不能完全杀死膜内细菌;膜内细菌在生物被膜环境中生长速率下降,有助于降低细菌对红霉素的敏感性.     

表皮葡萄球菌生物被膜对红霉素渗透性的研究

为探讨表皮葡萄球菌生物被膜对红霉素的渗透性, 我们采用生物被膜抗生素渗透模型检测Staphylcoccus epidermidis 1457、1457-msrA和临床分离株S68生物被膜不同时间点红霉素的渗透率, 并用吖啶橙染色激光共聚焦显微镜观察生物被膜内细菌RNA/DNA的相对含量; 扫描电子显微镜观察膜内细菌的密度。红霉素作用36 h后, 1457、1457-msrA、S68的渗透率分别为0.93, 0.55和 0.4; 1457渗透地较快, 8 h后渗透率即达到0.58, 而1457msrA和S68相对较为缓慢, 24 h后分别为0.499和0.31; 吖啶橙染色可见红霉素作用下膜内菌RNA和DNA的相对比例减小, 生长速率下降; 扫描电子显微镜观察可见生物被膜红霉素作用后空气面的细菌数与琼脂面相比均较少, 细胞碎片相对较多, 而对照组(无抗生素作用)琼脂面和空气面的细菌密度和分布较均匀。可见红霉素可渗透入表葡菌生物被膜, 但不能完全杀死膜内细菌; 膜内细菌在生物被膜环境中生长速率下降, 有助于降低细菌对红霉素的敏感性。     

The interaction of phage and biofilms

Biofilms present complex assemblies of micro-organisms attached to surfaces. they are dynamic structures in which various metabolic activities and interactions between the component cells occur. When phage come in contact with biofilms, further interactions occur dependent on the susceptibility of the biofilm bacteria to phage and to the availability of receptor sites. If the phage also possess polysaccharide-degrading enzymes, or if considerable cell lysis is effected by the phage, the integrity of the biofilm may rapidly be destroyed. Alternatively, coexistence between phage and host bacteria within the biofilm may develop. Although phage have been proposed as a means of destroying or controlling biofilms, the technology for this has not yet been successfully developed.     

生物被膜分散方式的研究进展

临床上生物被膜与感染的慢性迁延不愈密切相关,而生物被膜菌的分散又会造成感染的反复急性发作,给临床感染的有效控制带来很大困难。生物被膜菌的分散过程受到了遗传和环境等多因素的调控,主要是通过蜂式分散、块式分散和毯式分散3种形式来实现的。深入进行生物被膜基础研究对改变目前临床感染治疗的窘境有重大意义。     

阿萨希毛孢子菌生物膜构建及水杨酸干预策略研究

目的在医院内常用生物材料聚氯乙烯（PVC）表面构建阿萨希毛孢子菌的生物膜,评估该生物膜对几种临床抗真菌药物的耐药性,并观察水杨酸是否对阿萨希毛孢子菌生物膜的形成有干预作用。方法菌株鉴定采用API20CAUX并经PCR鉴定复核;使用PVC于RPM11640-MOPS中培养进行阿萨希毛孢子菌生物膜构建;MIC测定采用法国生物梅里埃公司ATB Fungus-3真菌药敏试剂条以及微量液体稀释法,并观察水杨酸对生物膜构建的影响。结果阿萨希毛孢子菌可以通过几个不连续阶段在聚氯乙烯表面形成生物膜,且已使用PVC块上附着的生物膜细胞比未使用PVC块上黏附的生物膜细胞明显密集;固着相即生物膜细胞的MIC比浮游相成倍提高;24h两性霉素B的MIC〉512μ/ml,且经两性霉素B的药物刺激后,阿萨希毛孢子明显可见芽管延长,菌丝交织;水杨酸作用后阿萨希毛孢子菌的菌丝明显变短,孢子短小。结论介入性器械可以作为阿萨希毛孢子菌生物膜构建的黏附基质,使微生物群体黏附于细胞外多聚材料表面而造成持续播散感染,因此生物膜干预对阿萨希毛孢子菌深部感染的治疗有很重要的意义。     

生物被膜主动分散机制研究进展

细菌生物被膜(bacterial biofilm, BBF)为微生物栖息提供了所需要的保护屏障和生长微环境。生物被膜对抗菌药物的耐受性使得它在医学治疗等领域产生了严重的危害。因此如何分散被膜显得意义重大。综述了生物被膜主动分散的几种主要机制,包括降解酶的合成、运动力的恢复、表面活性剂的产生和细胞死亡。     

Two-Fluid Model of Biofilm Disinfection

We consider a dynamic model of biofilm disinfection in two dimensions. The biofilm is treated as a viscous fluid immersed in a fluid of less viscosity. The bulk fluid moves due to an imposed external parabolic flow. The motion of the fluid is coupled to the biofilm inducing motion of the biofilm. Both the biofilm and the bulk fluid are dominated by viscous forces, hence the Reynolds number is negligible and the appropriate equations are Stokes equations. The governing partial differential equations are recast as boundary integral equations using a version of the Lorenz reciprocal relationship. This allows for robust treatment of the simplified fluid/biofilm motion. The transport of nutrients and antimicrobials, which depends directly on the velocities of the fluid and biofilm, is also included. Disinfection of the bacteria is considered under the assumption that the biofilm growth is overwhelmed by disinfection. Supported by NSF award DMS-0612467.     

The Use of Cellulase in Inhibiting Biofilm Formation from Organisms Commonly Found on Medical Implants

A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants. A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source. Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density. Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units ml^{m 1}. In addition, a control study using deactivated cellulase was performed. The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P. aeruginosa on glass surfaces. The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7. The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P. aeruginosa exopolysaccharides (EPS). The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme. An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P. aeruginosa. While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness.     

Indole Affects Biofilm Formation in Bacteria

Biofilm is bacterial population adherent to each other and to surfaces or interfaces, often enclosed by a matrix. Various biomolecules contribute to the establishment of biofilms, yet the process of building a biofilm is still under active investigation. Indole is known as a metabolite of amino acid tryptophan, which, however, has recently been proved to participate in various aspects of bacterial life including virulence induction, cell cycle regulation, acid resistance, and especially, signaling biofilm formation. Moreover, indole is also proposed to be a novel signal involved in quorum sensing, a bacterial cooperation behavior sometimes concerning the biofilm formation. Here the signaling role and molecular mechanism of indole on bacterial biofilm formation are reviewed, as well discussed is its relation to bacterial living adaptivity.     

Bacteriophages and Lysins in Biofilm Control

To formulate the optimal strategy of combatting bacterial biofilms, in this review we update current knowledge on the growing problem of biofilm formation and its resistance to antibiotics which has spurred the search for new strategies to deal with this complication. Based on recent findings, the role of bacteriophages in the prevention and elimination of biofilm-related infections has been emphasized. In vitro, ex vivo and in vivo biofilm treatment models with single bacteriophages or phage cocktails have been compared. A combined use of bacteriophages with antibiotics in vitro or in vivo confirms earlier reports of the synergistic effect of these agents in improving biofilm removal. Furthermore, studies on the application of phage-derived lysins in vitro, ex vivo or in vivo against biofilm-related infections are encouraging. The strategy of combined use of phage and antibiotics seems to be different from using lysins and antibiotics. These findings suggest that phages and lysins alone or in combination with antibiotics may be an efficient weapon against biofilm formation in vivo and ex vivo, which could be useful in formulating novel strategies to combat bacterial infections. Those findings proved to be relevant in the prevention and destruction of biofilms occurring during urinary tract infections,orthopedic implant-related infections, periodontal and peri-implant infections. In conclusion, it appears that most efficient strategy of eliminating biofilms involves phages or lysins in combination with antibiotics, but the optimal scheme of their administration requires further studies.     

营养及水力条件影响光合细菌生物膜生长特性实验

对平板式生物膜反应器内,流量及底物浓度范围分别为37.8~1080ml/h、0.05~10g/L的不同生长条件下光合产氢细菌生物膜生长特性进行了实验研究,讨论了不同水力及营养条件对沼泽红假单胞菌生物膜表面覆盖率、膜厚、干重和密度的影响。实验结果表明,不同水力及营养条件对生物膜生长速率及结构具有重要影响。在相同的时间间隔内,在高流速条件下光合细菌菌落生长较快,但过高的液体流速会导致部分生物膜脱落;高流速条件易使生物膜形成薄而致密的结构。光合细菌生物膜在循环液底物浓度较高时生长较快,密度也最高;而贫营养条件可以促成结构疏松生物膜在固液界面的形成,这种生物膜结构有利于微生物在低底物浓度条件下底物在生物膜内的传输。     

Polysaccharide-producing bacteria isolated from paper machine slime deposits

Development of novel enzymatic methods for slime deposit control in paper mills requires knowledge of polysaccharide-producing organisms and the polysaccharide structures present in deposits. In this work, 27 polysaccharide-producing bacteria were isolated from slime samples collected from different parts of a paper machine. Most of the isolates produced polysaccharides in liquid culture and nine of them were selected for production of polysaccharides for characterisation. The selected isolates belonged to seven different genera: Bacillus, Brevundimonas, Cytophaga, Enterobacter, Klebsiella, Paenibacillus and Starkeya. Using ribotyping, partial 16S rDNA sequencing, physiological tests and fatty acid analysis, four of the nine isolates: Bacillus cereus, Brevundimonas vesicularis, K. pneumoniae and P. stellifer were identified to the species level. Production of polysaccharides by the selected isolates varied between 0.07 and 1.20 g L^–1, the highest amount being produced by B. vesicularis. The polysaccharides were heteropolysaccharides with varying proportions of galactose, glucose mannose, rhamnose fucose and uronic acids.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

嗜水气单胞菌生物被膜对其耐药性的影响

建立了嗜水气单胞菌(Aeromonas hydrophila, Ah)的生物被膜(Bacterial biofilm, BF) 体外形成模型, 并对11种抗菌药物对BF细菌和浮游（Freecell, FC）细菌的清除作用进行了研究。将Ah J1株在放有硅胶膜的TSB中培养7d,用银染法鉴定,发现可形成良好的BF。FC细菌对青霉素具有耐药性, 最低杀菌浓度(MBC)为256μg/mL;对蒽诺沙星和氟哌酸最敏感,MBC分别为003μg/mL和0.25μg/mL。氟苯尼考对BF细菌的清除能力最强,作用于BF细菌和FC细菌的MBC之比为2∶1;卡那霉素、青霉素、新霉素的MBC比值在32∶1以上。扫描电镜观察蒽诺沙星作用于FC及BF细菌前后的形态变化,并测定其杀菌曲线。发现4×MBC时可完全清除FC细菌,但不能完全清除BF细菌;在32×MBC时,4h内可完全清除FC细菌,而24h内完全清除BF细菌。结果表明形成BF的Ah对抗菌药物可形成强耐受性,其潜在影响应引起足够重视。     

引发医院感染表皮葡萄球菌生物被膜的检测

为了解引发医院感染的表皮葡萄球菌中ica操纵元的存在与生物被膜的产生的关系及其对抗生素敏感性的影响,收集了引发医院感染的表葡萄球菌106株,采用定量和定性法检测生物被膜的产生,PCR法检测ica操纵元基因的存在以及测量细菌对红霉素（ERY）、氨苄青霉素（AMP）、头孢西丁（FOX）、头孢曲松（CRO）、替考拉宁（TEC）、环丙沙星（CIP）、四环素（TCY）、复方新诺明（SXT）、万古霉素（VAN）的最小抑菌浓度（MIC）;106株表皮葡萄球菌分离株中,有33株检测出icaABC（31．1％）;ica^＋菌中产膜菌的检出率高于ica^＋菌（P=0．001）;葡萄糖和NaCl可提高产膜菌的检出率;ica^＋浮游菌对红霉素,头孢西丁和头孢曲松的耐药率高于ica^＋浮游菌株,但对氨苄青霉素,环丙沙星,四环素和复方新诺明的耐药率与ica^＋菌相似;ica位点基因的存在与引发表葡菌医院感染密切相关,但生物被膜内菌耐药机制还需进一步研究。     

Biofilm formation in water cooling systems

Biofilm formation on stainless steel samples immersed in cooling water has been evaluated by exposing metal samples to cooling seawater for 30 days. Anaerobic bacteria were then at 1.6 × 10⁶/cm², with sulphate-reducing species predominating. Aerobic bacteria and fungi were 2600 and 140/cm², respectively. After 60 days, numbers of aerobic microorganisms remained constant whereas the count of anaerobic microorganisms had increased to 1.8×10⁹/cm². Scanning electron microscopy showed the presence of morphologically different microorganisms in deposits and as a mucilaginous net. No signs of corrosion were detected on the stainless steel surface.The authors are with the Departamento de Engenharia Bioquimica Centro de Tecnologia, Bloco E. Universidade Federal do Rio de Janeiro Ilha do Fundão, 21941-900 Rio de Janeiro, Brazil     

Isolation,Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.     

Iron Oxidation and Deposition in the Biofilm Covering Montacuta ferruginosa (Mollusca,Bivalvia)

The shell of the bivalve Montacuta ferruginosa is covered with a rust-colored biofilm. This biofilm includes filamentous bacteria and protozoa encrusted with a mineral, rich in ferric ion and phosphate. The aim of this research was to study two possible microbial iron precipitation pathways in the biofilm, namely, microbial iron oxidation and microbial degradation of organic Fe(III) complexes. The iron-oxidizing activity was assayed spectrophotometrically by monitoring the formation of the dye Wurster blue in biofilm extracts. Iron-oxidizing activity was effectively detected in extracts obtained by oxalic acid treatment of biofilm fragments. Extracts obtained without oxalic acid treatment, heated extracts, or extracts supplemented with HgCl 2 did not show any activity. This suggests that an iron-oxidizing factor (IOF), possibly an enzyme, coprecipitated with the mineral. Additional information gathered by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel-filtration chromatography, and UV spectrophotometry indicate that the IOF would be a small peptide or glycopeptide (1,350 Da). Microbial degradation of organic Fe(III) complexes was assayed with biofilm fragments incubated in a medium containing ferric citrate. Analysis of the supernatants after various intervals revealed that the complex was degraded by living microorganisms much faster than in the heat-killed negative controls. We conclude that ferric iron precipitation in the biofilm may proceed by way of microbial Fe(II) oxidation as well as microbial degradation of organic Fe(III) complexes.