材料表面特征对生物膜形成的影响及其应用

生物膜是微生物细胞粘附于材料表面的群体性生长方式。在实践应用中,有目的地调控微生物在材料表面的成膜进程具有重要意义。本文概述了生物膜在材料表面的形成机制及其影响因素,综述了材料表面的电荷特征、亲疏水性、形貌模式和功能性化学修饰等物化特性对细胞粘附和生物膜形成的影响,并介绍了目前在不同实际应用场景中抑制成膜和促进成膜材料的研发现状。     

The impact of medicinal brines on microbial biofilm formation on inhalation equipment surfaces

Abstract

Materials such as polyvinyl chloride, polypropylene, and polyethylene are used for the construction of medical equipment, including inhalation equipment. Inhalation equipment, because of the wet conditions and good oxygenation, constitutes a perfect environment for microbial biofilm formation. Biofilms may affect microbiological cleanliness of inhalation facilities and installations and promote the development of pathogenic bacteria. Microbial biofilms can form even in saline environments. Therefore, the aim of this study was to evaluate the effect of medicinal brines on microbial biofilm formation on the surfaces of inhalation equipment. The study confirmed the high risk of biofilm formation on surfaces used in inhalation equipment. Isolated microorganisms belonged to potential pathogens of the respiratory system, which can pose a health threat to hospital patients. The introduction of additional contaminants increased the amount of bacterial biofilm. On the other hand, the presence of brines significantly limited the amount of biofilm, thus eliminating the risk of infections.     

Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis

Bacterial biofilm formation on contact lenses (CLs), and CL storage cases may be a risk factor for CL-associated corneal infection and may explain the persistence of organisms in CL storage cases. This study evaluated biofilm formation on, and microbial contamination of, CLs and CL storage cases from patients with microbial keratitis. Contact lenses and CL storage cases from 20 wearers with microbial keratitis were sampled microbiologically and visualized using scanning electron microscopy (SEM). Culture results from the cornea were also noted. Bacterial biofilm was present more frequently ( P < 0·05) on CL storage case surfaces (17/20) compared with CL surfaces (11/20) and biofilm density was significantly greater on case surfaces ( P < 0·05). There was no association between poor compliance and microbial contamination of the CL storage case, nor between poor compliance and biofilm formation or density on the CL or CL storage case. Biofilm formation occurred equally frequently with hydrogen peroxide and chlorine release care systems. Microbial keratitis in CL wearers is frequently associated with bacterial biofilm in the CL storage case. Despite the use of current CL disinfection systems, the CL storage case is a favourable environment for proliferation of certain organisms. Biofilm on CLs may prolong the retention time of organisms at the ocular surface and increase their potential pathogenicity.     

Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C. At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Chlorination-mediated EPS excretion shapes early-stage biofilm formation in drinking water systems

Microbial surface adhesion to surfaces and subsequent biofilm establishment are ubiquitous in drinking water systems, which often contribute to deteriorated water quality. Disinfectants are common agents applied to drinking water controlling microbial propagation, yet the underlying mechanisms of how disinfectants function to regulate microbial activity and thereby biofilm development remains elusive. We experimentally studied the effects of chlorination on extracellular polymeric substance (EPS) production, and its impacts on early-stage biofilm formation in a model drinking water system. Results showed that low-level chlorine (≤ 1.0 mg/L) stimulated microbial EPS (especially of proteins) excretion that favored early-stage biofilm formation. Microbes experiencing higher chlorination (>1.0 mg/L) exhibited clearly suppressed growth associated with reduced EPS release, consequently yielding less biofilm formation. Removal of cell-attached proteins and polysaccharides diminished biofilm formation, which highlighted the critical role of EPS (especially protein components) in biofilm development. A negative correlation between chlorination-mediated microbial protein production and cell surface charge suggested that chlorine disinfection may modify cell surface properties through regulation of microbial EPS excretion and thereby mediate biofilm formation. With these quantitative estimations, this study provides novel insights into how chlorination-mediated EPS excretion shapes early-stage biofilm formation, which is essential for practical functioning of drinking water systems.     

Adhesion as a weapon in microbial competition

Microbes attach to surfaces and form dense communities known as biofilms, which are central to how microbes live and influence humans. The key defining feature of biofilms is adhesion, whereby cells attach to one another and to surfaces, via attachment factors and extracellular polymers. While adhesion is known to be important for the initial stages of biofilm formation, its function within biofilm communities has not been studied. Here we utilise an individual-based model of microbial groups to study the evolution of adhesion. While adhering to a surface can enable cells to remain in a biofilm, consideration of within-biofilm competition reveals a potential cost to adhesion: immobility. Highly adhesive cells that are resistant to movement face being buried and starved at the base of the biofilm. However, we find that when growth occurs at the base of a biofilm, adhesion allows cells to capture substratum territory and force less adhesive, competing cells out of the system. This process may be particularly important when cells grow on a host epithelial surface. We test the predictions of our model using the enteric pathogen Vibrio cholerae, which produces an extracellular matrix important for biofilm formation. Flow cell experiments indicate that matrix-secreting cells are highly adhesive and form expanding clusters that remove non-secreting cells from the population, as predicted by our simulations. Our study shows how simple physical properties, such as adhesion, can be critical to understanding evolution and competition within microbial communities.     

口腔种植材料表面特性与生物膜形成研究进展

人类口腔环境为微生物提供了适宜生存的条件,多种微生物在牙齿表面形成了由基质包裹相互粘附的口腔生物膜,口腔生物膜是口腔微生物生存、代谢和致病的基础。随着1965年Brånemark种植体在临床上的成功应用,种植相关材料周围致病菌导致的种植体周围炎成为种植修复最常见的并发症之一,影响种植修复的远期效果。种植体周围炎引起了许多关注,并且口腔种植材料表面的特性和口腔生物膜的形成密切相关。本文就种植材料及天然牙齿表面的生物膜形成、种植材料表面特性对口腔生物膜及细菌粘附的影响因素、增强种植材料抗菌性能的方法以及未来的研究方向等作一综述。     

白假丝酵母菌生物膜的研究进展

随着医疗水平的不断发展,越来越多的医疗操作、医疗设备和药物可能导致人体正常的微生物平衡被打破,使得机会致病菌白假丝酵母菌的感染呈现逐年上升的趋势。白假丝酵母菌在宿主或医疗器械表面形成生物膜的能力是一个十分关键的毒力因素。生物膜可以帮助白假丝酵母菌成功逃避宿主免疫并产生较强的耐药性,从而导致难治性真菌感染。本文从白假丝酵母菌生物膜的形成过程、生物膜相关的主要基因和影响生物膜毒力的因素3个方面介绍近年来的研究进展,为进一步研究白假丝酵母菌生物膜的形成机制提供参考。     

Bacterial colonization and biofilm development on minimally processed vegetables

Bacterial biofilms have been observed and reported on food and food-processing surfaces and can contribute to increased risks for product quality and food safety. The colonization of fruit and vegetables by pectynolitic bacteria like Pseudonomas fluorescens attributable to conditions such as soft rot, can also manifest as biofilms. A developed biofilm structure can provide a protective environment for pathogens such as Listeria monocytogenes reducing the effectiveness of sanitisers and other inhibitory agents. Understanding the colonization of bacteria on leaf surfaces is essential to the development of a better understanding of the leaf ecology of vegetable products. Studies of microbial colonization of leaf surfaces have been conducted using SEM and more recently using confocal microsocpy techniques. In the current study, a Leica TCS NT laser scanning confocal microscope was used to investigate biofilm formation using vital fluorescence staining on intact vegetable leaves. Reflection contrast and fluorescence three-dimensional imaging successfully delineated bacterial and biofilm morphology without disturbing the bacterial or leaf surface structure. The results demonstrate the presence and development of biofilm on the surface of lettuce. The biofilms appeared to originate on the cuticle in distinct micro-environments such as in the natural depression of the stomata, or in the intercellular junction. Bacteria also adhered to and developed biofilm colonies within an hour of contact and with clean stainless steel surfaces. Our study investigates the progression of biofilm formation from leaf colonization, and will assist in characterising the critical mechanisms of plant/host interaction and facilitate the development of improved preservation, sanitising and packaging strategies for minimally processed vegetable products.     

Listeria monocytogenes LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Effect of a synbiotic on microbial community structure in a continuous culture model of the gastric microbiota in enteral nutrition patients

Patients with dysphagia require long-term nutritional support. This can be delivered by the enteral route via a percutaneous endoscopic gastrostomy (PEG) tube. Enteral nutrition (EN) bypasses the body's innate defences that prevent the microbial colonization of the proximal gut, which predisposes to microbial overgrowth. A continuous culture model simulating the upper gastrointestinal tract microbiota of EN patients was used to investigate the effects of a synbiotic (Lactobacillus acidophilus DUN-311, Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01, Synergy 1) on microbial community structure and metabolism. A PEG tube was inserted into the fermenters to study biofilm formation. The synbiotic delivered in sterile semi-skimmed milk (SSSM) was introduced either 48?h prior to or after PEG tube insertion. The synbiotic reduced biofilm formation on PEG tube surfaces, with suppression of Escherichia coli and Klebsiella pneumoniae when it was added subsequent to PEG insertion. When synbiotic feeding was commenced prior to PEG insertion, colonization by Staphylococcus aureus, Candida albicans and Candida famata was also inhibited. Lactate production increased in response the synbiotic or control (SSSM). These results indicate that the use of a synbiotic has the potential to reduce pathogen colonization on PEG tube surfaces in vivo, thereby reducing the incidence of biofilm-related infectious complications.     

Bacterial biofilms and surface fouling

Bacteria are attracted to surfaces. Their surface adhesion, with subsequent binary fission and exopolymer production, leads to the formation of biofilms. Such biofilms consist of bacterial cells in a matrix of their own exopolysaccharide glycocalyces. In addition to the bulk fluid and the surface, biofilms constitute a third physical phase. The close proximity of the bacterial cells in the biofilm matrices assists the formation of metabolically dependent consortia. The chemical and physical activities of these microbial communities produces a heterogeneous system at the colonised surface. Metabolites, produced at specific points on the surface, can lead to the development of effective anodes and cathodes at adjoining locations on the surface. In this way the fouling of a surface by bacterial biofilm development facilitates focal attack on that surface. This pit formation is characteristic of bacterial surface activities as diverse as dental decay and metal corrosion. In this review, we examine bacterial adhesion, biofilm formation and several instances of focal bacterial attack on colonised surfaces. However, pathogenic biofilms and the fouling of biological surfaces, with the exception of caries formation, is outside the scope of this paper.     

Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10(+) and Asc10(-)E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2-4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis.     

Microbe-surface interactions in biofouling and biocorrosion processes.

The presence of microorganisms on material surfaces can have a profound effect on materials performance. Surface-associated microbial growth, i.e. a biofilm, is known to instigate biofouling. The presence of biofilms may promote interfacial physico-chemical reactions that are not favored under abiotic conditions. In the case of metallic materials, undesirable changes in material properties due to a biofilm (or a biofouling layer) are referred to as biocorrosion or microbially influenced corrosion (MIC). Biofouling and biocorrosion occur in aquatic and terrestrial habitats varying in nutrient content, temperature, pressure and pH. Interfacial chemistry in such systems reflects a wide variety of physiological activities carried out by diverse microbial populations thriving within biofilms. Biocorrosion can be viewed as a consequence of coupled biological and abiotic electron-transfer reactions, i.e. redox reactions of metals, enabled by microbial ecology. Microbially produced extracellular polymeric substances (EPS), which comprise different macromolecules, mediate initial cell adhesion to the material surface and constitute a biofilm matrix. Despite their unquestionable importance in biofilm development, the extent to which EPS contribute to biocorrosion is not well-understood. This review offers a current perspective on material/microbe interactions pertinent to biocorrosion and biofouling, with EPS as a focal point, while emphasizing the role atomic force spectroscopy and mass spectrometry techniques can play in elucidating such interactions.     

Microbe repelling coated stainless steel analysed by field emission scanning electron microscopy and physicochemical methods

Coating of stainless steel with diamond-like carbon or certain fluoropolymers reduced or almost eliminated adhesion and biofilm growth of Staphylococcus epidermidis, Deinococcus geothermalis, Meiothermus silvanus and Pseudoxanthomonas taiwanensis. These species are known to be pertinent biofilm formers on medical implants or in the wet-end of paper machines. Field emission scanning electron microscopic analysis showed that Staph. epidermidis, D. geothermalis and M. silvanus grew on stainless steel using thread-like organelles for adhesion and biofilm formation. The adhesion threads were fewer in number on fluoropolymer-coated steel than on plain steel and absent when the same strains were grown in liquid culture. Psx. taiwanensis adhered to the same surfaces by a mechanism involving cell ghosts on which the biofilm of live cells grew. Hydrophilic (diamond-like carbon) or hydrophobic (fluoropolymer) coatings reduced the adherence of the four test bacteria on different steels. Selected topographic parameters, including root-mean-square roughness (S (q)), skewness (S (sk)) and surface kurtosis (S (ku)), were analysed by atomic force microscopy. The surfaces that best repelled microbial adhesion of the tested bacteria had higher skewness values than those only slightly repelling. Water contact angle, measured (theta (m)) or roughness corrected (theta (y)), affected the tendency for biofilm growth in a different manner for the four test bacteria.     

Biofilm-defective mutants of Bacillus subtilis

Many bacteria can adopt organized, sessile, communal lifestyles. The gram-positive bacterium, Bacillus subtilis,forms biofilms on solid surfaces and at air-liquid interfaces, and biofilm development is dependent on environmental conditions. We demonstrate that biofilm formation by B. subtilis strain JH642 can be either activated or repressed by glucose, depending on the growth medium used, and that these glucose effects are at least in part mediated by the catabolite control protein, CcpA. Starting with a chromosomal Tn917-LTV3 insertional library, we isolated mutants that are defective for biofilm formation. The biofilm defects of these mutants were observable in both rich and minimal media, and both on polyvinylchloride abiotic surfaces and in borosilicate tubes. Two mutants were defective in flagellar synthesis. Chemotaxis was shown to be less important for biofilm formation than was flagellar-driven motility. Although motility is known to be required for biofilm formation in other bacteria, this had not previously been demonstrated for B. subtilis. In addition, our study suggests roles for glutamate synthase, GltAB, and an aminopeptidase, AmpS. The loss of these enzymes did not decrease growth or cellular motility but had dramatic effects on biofilm formation under all conditions assayed. The effect of the gltAB defect on biofilm formation could not be due to a decrease in poly-gamma-glutamate synthesis since this polymer proved to be nonessential for robust biofilm formation. High exogenous concentrations of glutamate, aspartate, glutamine or proline did not override the glutamate synthase requirement. This is the first report showing that glutamate synthase and a cytoplasmic aminopeptidase play roles in bacterial biofilm formation. Possible mechanistic implications and potential roles of biofilm formation in other developmental processes are discussed.     

Signals,Regulatory Networks,and Materials That Build and Break Bacterial Biofilms

Summary: Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.     

Alumina surfaces with nanoscale topography reduce attachment and biofilm formation by Escherichia coli and Listeria spp.

This work reports on a simple, robust and scientifically sound method to develop surfaces able to reduce microbial attachment and biofilm development, with possible applications in medicine, dentistry, food processing, or water treatment. Anodic surfaces with cylindrical nanopores 15 to 100 nm in diameter were manufactured and incubated with Escherichia coli ATCC 25922 and Listeria innocua. Surfaces with 15 and 25 nm pore diameters significantly repressed attachment and biofilm formation. Surface–bacteria interaction forces calculated using the extended Derjaguin Landau Verwey-Overbeek (XDLVO) theory indicate that reduction in attachment and biofilm formation is due to a synergy between electrostatic repulsion and surface effective free energy. An attachment study using E. coli K12 strains unable to express appendages also suggests that the small-pore surfaces may inhibit flagella-dependent attachment. These results can have immediate, far-reaching implications and commercial applications, with substantial benefits for human health and life.     

SdrC induces staphylococcal biofilm formation through a homophilic interaction

The molecular pathogenesis of many Staphylococcus aureus infections involves growth of bacteria as biofilm. In addition to polysaccharide intercellular adhesin (PIA) and extracellular DNA, surface proteins appear to mediate the transition of bacteria from planktonic growth to sessile lifestyle as well as biofilm growth, and can enable these processes even in the absence of PIA expression. However, the molecular mechanisms by which surface proteins contribute to biofilm formation are incompletely understood. Here we demonstrate that self‐association of the serine‐aspartate repeat protein SdrC promotes both bacterial adherence to surfaces and biofilm formation. However, this homophilic interaction is not required for the attachment of bacteria to abiotic surfaces. We identified the subdomain that mediates SdrC dimerization and subsequent cell‐cell interactions. In addition, we determined that two adjacently located amino acid sequences within this subdomain are required for the SdrC homophilic interaction. Comparative amino acid sequence analysis indicated that these binding sites are conserved. In summary, our study identifies SdrC as a novel molecular determinant in staphylococcal biofilm formation and describes the mechanism responsible for intercellular interactions. Furthermore, these findings contribute to a growing body of evidence suggesting that homophilic interactions between surface proteins present on neighbouring bacteria induce biofilm growth.