空肠弯曲杆菌生物膜形成和调控机理研究进展北大核心CSCD

空肠弯曲杆菌(Campylobacter jejuni)分泌胞外多糖和各种胞外蛋白和核酸等相互交联在一起构成生物膜,可增强其在不利环境下的生存率,尤其是对各种洗涤剂、抗生素和消毒剂的耐受力。本文从介质表面性质、温度、气体环境、以及基因的调控等多方面阐述了空肠弯曲杆菌生物膜结构及形成调控机制,同时对各种去除生物膜的实际应用做了分析和展望,为探寻生物膜的控制方法提供参考。     

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions

The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.     

Effects of phenol and natural phenolic compounds on biofilm formation by Pseudomonas aeruginosa

A biofilm is formed as a result of adhesion of microorganisms to various surfaces with the production of extracellular polymers (polysaccharides and proteins). Biofilms cause serious problems in the chemical, medical and pharmaceutical industries. Recent findings indicate that some natural phenolic compounds found in plants have an anti-biofouling effect on biofilm formation by Gram-negative bacteria. The anti-biofouling activities of 14 selected phenol and natural phenolic compounds were tested against Pseudomonas aeruginosa, using a microtiter-plate. A modified microtiter-plate assay was used because it enabled indirect measurement of bacterial cells attached to the surface of the wells. This assay involved fixing the bacterial film with methanol, staining with crystal violet dye and then releasing the bound dye with 33% glacial acetic acid. The optical density (OD) of the solution was measured at 570 nm by using an automated ICN Flow Titertek Multiscan Plus reader. Phenol and natural phenolic compounds except ethyl linoleate and tocopherol showed a significant reduction in biofilm formation by P. aeruginosa.     

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.     

副溶血性弧菌-霍乱弧菌混合生物被膜形成过程研究

【目的】研究副溶血性弧菌(Vibrioparahaemolyticus,VP)和霍乱弧菌(Vibriocholera,VC)混合生物被膜的形成过程。【方法】在4、8、12、24、36、48、60、72 h测定单独条件下VP、VC及其混合后生物被膜的形成情况,通过结晶紫染色法、平板菌落计数法、测定胞外多糖、胞外蛋白,通过荧光原位杂交(FISH)观察混合生物被膜形成。【结果】虽然形成的混合生物被膜量介于VC和VP之间,但混合生物被膜在形成过程中,成熟期后生物被膜量的变化较小,对环境的抗性增强。混合生物被膜中拥有更多的活菌,混合生物被膜形成过程中胞外蛋白和胞外多糖的变化体现出其可能在对抵御不适应环境中起重要作用,通过FISH可观察到不同时期生物被膜的变化过程。【结论】VC与VP共同形成生物被膜的过程中,混合生物被膜总量虽然减少,但混合生物被膜中拥有更多的活菌,这可能引起更大的危害。研究混合生物被膜形成过程中被膜的变化,可为有害生物被膜的控制提供基础。     

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis‐enriched floccular and granular biofilm

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory‐scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high‐stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter‐enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.     

Proteomic analysis of cytoplasmic and surface proteins from yeast cells,hyphae, and biofilms of Candida albicans

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non‐covalently attached cell‐surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (> 1.5‐fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi‐organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.     

基于高通量共聚焦激光扫描显微镜方法定量分析副溶血性弧菌生物被膜结构

【目的】副溶血性弧菌是水产品中常见的食源性致病菌,生物被膜的形成对副溶血性弧菌的环境生存和传播至关重要。这项工作的目的是评估临床和环境中分离出的44株副溶血性弧菌菌株形成的生物被膜的结构多样性。【方法】该研究基于共聚焦激光扫描显微镜的高通量方法,使用与高分辨率成像兼容的96孔微量滴定板,结合结构分析软件ISA-2来研究生物被膜形成和结构,分析22株食品与22株临床来源的副溶血性弧菌菌株形成的生物被膜结构参数(生物体积、平均厚度、粗糙系数)。【结果】CLSM图像显示,44株副溶血性弧菌菌株在培养48h后能够形成3D结构,进一步比较分析了临床来源菌株与环境来源菌株形成的生物被膜结构异同,发现临床菌株生物被膜的变异系数比环境菌株生物被膜的变异系数小,且同时携带tdh和trh两种毒力因子的菌株生物被膜变异性最小。凝聚层次聚类分析结果显示,副溶血性弧菌生物被膜可以分为致密且表面光滑(39%)、斑驳且表面粗糙(27%)、疏松且表面坑洼(34%),临床菌株易形成致密且表面光滑和斑驳且表面粗糙的生物被膜,而环境菌株易形成致密且表面光滑和疏松且表面坑洼的生物被膜。【结论】该研究深入了解了副溶血性弧菌生物...     

Surface properties,adhesion and biofilm formation on different surfaces by Scedosporium spp. and Lomentospora prolificans

Abstract

In the present work, some surface properties of the fungi Scedosporium apiospermum, S. aurantiacum, S. minutisporum, and Lomentospora prolificans and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4?h of fungi–surface contact. Additionally, after 72?h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72?h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that Scedosporium/Lomentospora spp. are capable of forming biofilms on diverse abiotic surfaces.     

Anti-adhesion activity of two biosurfactants produced by <Emphasis Type="Italic">Bacillus</Emphasis> spp. prevents biofilm formation of human bacterial pathogens

In this work, two biosurfactant-producing strains, Bacillus subtilis and Bacillus licheniformis, have been characterized. Both strains were able to grow at high salinity conditions and produce biosurfactants up to 10% NaCl. Both extracted-enriched biosurfactants showed good surface tension reduction of water, from 72 to 26–30 mN/m, low critical micelle concentration, and high resistance to pH and salinity. The potential of the two lipopeptide biosurfactants at inhibiting biofilm adhesion of pathogenic bacteria was demonstrated by using the MBEC device. The two biosurfactants showed interesting specific anti-adhesion activity being able to inhibit selectively biofilm formation of two pathogenic strains. In particular, Escherichia coli CFT073 and Staphylococcus aureus ATCC 29213 biofilm formation was decreased of 97% and 90%, respectively. The V9T14 biosurfactant active on the Gram-negative strain was ineffective against the Gram-positive and the opposite for the V19T21. This activity was observed either by coating the polystyrene surface or by adding the biosurfactant to the inoculum. Two fractions from each purified biosurfactant, obtained by flash chromatography, fractions (I) and (II), showed that fraction (II), belonging to fengycin-like family, was responsible for the anti-adhesion activity against biofilm of both strains.     

A dynamic in vitro model for evaluating antimicrobial activity against bacterial biofilms using a new device and clinical-used catheters

The activity of daptomycin compared to vancomycin against Staphylococcus epidermidis-biofilms on intravascular catheters has been evaluated using the new Sevilla device that enables to use medical grade-catheters, in an in vitro model that simulates the in vivo conditions. S. epidermidis-biofilms were obtained on polyurethane catheter segments using the Sevilla device linked to a continuous culture system for 24 h. To assess the antimicrobial activity, at this time the continuous culture system was changed to therapeutic antimicrobial concentration solutions for 48 h. At each 24 h interval time, catheter segments were taken out, washed and sonicated. Viable adherent bacteria were determined by agar plating. Data of surviving bacteria numbers attached to the catheter surface obtained with the Sevilla device showed a very good reproducibility. Daptomycin showed a good activity against S. epidermidis-biofilm on polyurethane catheter surface. After 48 h exposure to daptomycin, surviving adherent bacteria were reduced by 4 log compared to the control with no antimicrobial. Using the same model, vancomycin reduced bacterial survival by only 1.3 log. The Sevilla device enables antimicrobial agent activity against bacterial biofilms grown on the external surface of catheters used in clinical practice to be evaluated. The model used replicates as closely as possible the biofilm formed in a highly standardized way. Using this model, daptomycin demonstrates potent in vitro activity against S. epidermidis-biofilm on a polyurethane catheter; this activity was greater than that showed by vancomycin.     

Evaluation of the kinetics and mechanism of action of anti‐integration host factor‐mediated disruption of bacterial biofilms

The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice‐like network wherein a member of the DNABII family of DNA‐binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using non‐typeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF‐specific and mediated by binding of transiently dissociated IHF by anti‐IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA‐binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.     

藤椒精油对腐败解淀粉芽孢杆菌DY1a生物被膜的抑制作用

【背景】芽孢杆菌是豆制品的重要腐败菌,在气液界面形成生物膜,对产品生产带来持续污染。【目的】探讨藤椒精油(Zanthoxylum armatum DC.essential oil,ZA-EO)对腐败解淀粉芽孢杆菌DY1a菌体及生物被膜的抑制作用与机制。【方法】采用气相色谱-质谱(gas chromatography-mass spectrometer,GC-MS)分析藤椒精油主要成分与相对含量,通过二倍稀释法测定藤椒精油对菌株的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC),并分析精油对腐败菌胞外蛋白酶活性、腐败菌生物被膜形成抑制及成熟生物被膜的清除作用,采用扫描电镜结合三维光学显微镜分析生物被膜形貌结构变化,测定生物被膜胞外聚合物(extracellular polymeric substance,EPS)多糖与蛋白质含量变化;并通过细菌运动能力、细胞黏附及自聚集能力、细胞表面疏水性和Zeta电位来初步探讨藤椒精油对生物被膜的抑制机理。【结果】藤椒精...     

Detection of Pseudomonas aeruginosa biofilm on medical biomaterials

The adhesion and biofilm formation of Pseudomonas aeruginosa strains on the surface of catheters made of various polymers (PU, SL, PCW) were determined in vitro. It was used the method by Richards et al. with modification of Rózalska et al. (1998), in which soluble colourless TTC is reduced to insoluble red formazan. The results of this study indicate that 80.3% of this strains adhered and 94.6% formed biofilm on the Nelaton catheter, 86% strains adhered and 76.1% formed biofilm on the polyurethane catheter, and 73.2% strains adhered, and 78.9% formed biofilm on the Foley catheter.     

细菌生物被膜基质的研究进展

细菌生物被膜(biofilm)附着在生物或者非生物表面,由细菌及其分泌的糖、蛋白质和核酸等多种基质组成的细菌群落,是造成病原细菌持续性感染、毒力和耐药性的重要原因之一.细菌的生物被膜基质由复杂的胞外聚合物(extracellular polymeric substances,EPS)构成,影响生物被膜的结构和功能.本文...     

Effect of Different Concentrations of Ortho-phthalaldehyde on Biofilms Formed by Pseudomonas fluorescens Under Different Flow Conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

Biofilms formed by Scedosporium and Lomentospora species: focus on the extracellular matrix

Abstract

In the present study, the composition of the extracellular matrix (ECM) of the biofilm formed by Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans on a polystyrene surface was investigated. Confocal laser scanning microscopy revealed a dense mycelial mass, with an ECM covering/interspersing the fungal cells and containing carbohydrate-rich molecules (e.g. glycoproteins) and extracellular DNA. The ECMs that were chemically extracted from mature biofilms formed by each of these fungi was predominantly composed of polysaccharides, followed by proteins, nucleic acids and sterols. In general, the amount of biofilm ECM was significantly greater in S. minutisporum and S. aurantiacum than in S. apiospermum and L. prolificans. Corroborating these results, the disarticulation of mature biofilms with enzymes, sodium metaperiodate and chelating agents occurred mainly in S. minutisporum and S. aurantiacum. Collectively, these results have revealed for the first time the composition of the ECM of the biofilms formed by Scedosporium/Lomentospora species and the role it plays in their architecture.     

Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo

Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA) has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR). Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.