Statistical evaluation of a newly modified Robbins device using a bioluminescent pseudomonad to quantify adhesion to plastic

A modified Robbins device (MRD) has frequently been used as a model system to study adhesion and biofilm formation. This study investigates the reproducibility of attachment and whether a statistically significant gradient of adhesion exists along the 25 sampling ports of a MRD. A simple, quantitative, non‐destructive, bioluminescence assay was developed in order to measure attachment of bioluminescent P. veronii BL146bio cells to plastic discs of Thermanox? in newly modified Robbins devices (nMRD). No statistically significant difference in mean bioluminescence values occurred between pairs of nMRDs run in parallel, but there was a significant difference in bioluminescence values between different batches of bacteria (p < 0.05). Generalised Linear Modelling showed that the position of the sample disc influenced the numbers attaching. In 50% of devices a significant positive gradient of attachment occurred and bioluminescence values varied from disc 1 to disc 25 by 29.6–58.0%. In the other 50% of nMRDs there was a smaller, non‐significant gradient. A disc sampling regime was devised to take this gradient into account and used to prove a positive correlation between bioluminescence and numbers of viable P. veronii BL146bio cells during a 6h biofilm accumulation period.     

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.     

Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics

The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Adhesion to and biofilm formation on IB3-1 bronchial cells by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates from cystic fibrosis patients

Background  

Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.     

Influence of surface copper content on Stenotrophomonas maltophilia biofilm control using chlorine and mechanical stress

Abstract

This work aimed to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. Stenotrophomonas maltophilia isolated from drinking water was used as a model microorganism and biofilms were developed in a rotating cylinder reactor using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10?mg l^?1 of free chlorine for 10?min, an increased shear stress (a fluid velocity of 1.5?m s^?1 for 30s), and a combination of both treatments. These shock treatments were not effective in biofilm control. The benefits from the use of copper surfaces was found essentially in reducing the numbers of non-damaged cells. Copper materials demonstrated better performance in biofilm prevention than chlorine. In general, copper alloys may have a positive public health impact by reducing the number of non-damaged cells in the water delivered after chlorine exposure.     

大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。     

Biofilm formation on the surface of monazite and xenotime during bioleaching

Microbial attachment and biofilm formation is a ubiquitous behaviour of microorganisms and is the most crucial prerequisite of contact bioleaching. Monazite and xenotime are two commercially exploitable minerals containing rare earth elements (REEs). Bioleaching using phosphate solubilizing microorganisms is a green biotechnological approach for the extraction of REEs. In this study, microbial attachment and biofilm formation of Klebsiella aerogenes ATCC 13048 on the surface of these minerals were investigated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In a batch culture system, K. aerogenes was able to attach and form biofilms on the surface of three phosphate minerals. The microscopy records showed three distinctive stages of biofilm development for K. aerogenes commencing with initial attachment to the surface occurring in the first minutes of microbial inoculation. This was followed by colonization of the surface and formation of a mature biofilm as the second distinguishable stage, with progression to dispersion as the final stage. The biofilm had a thin-layer structure. The colonization and biofilm formation were localized toward physical surface imperfections such as cracks, pits, grooves and dents. In comparison to monazite and xenotime crystals, a higher proportion of the surface of the high-grade monazite ore was covered by biofilm which could be due to its higher surface roughness. No selective attachment or colonization toward specific mineralogy or chemical composition of the minerals was detected. Finally, in contrast to abiotic leaching of control samples, microbial activity resulted in extensive microbial erosion on the high-grade monazite ore.     

Characterisation and in vitro activities of surface attached dihydropyrrol-2-ones against Gram-negative and Gram-positive bacteria

Bacterial infection of biomedical devices is still a major barrier to their use. This is compounded by increasing antibiotic resistance. Here, the specific covalent attachment of a series of dihydropyrrol-2-one (DHP), analogues of bacterial quorum sensing inhibitors, to surfaces via a Michael-type addition reaction is described. Differences in efficiency of attachment related to the substituent groups were found by X-ray photoelectron spectroscopy. The physical characteristics of the surfaces were further explored by atomic force microscopy and contact angle measurements. The ability of these coatings to prevent the formation of a biofilm by Pseudomonas aeruginosa and Staphylococcus aureus was examined using confocal laser scanning microscopy and image analysis. The DHP-treated surfaces showed significant reductions in bacterial adhesion without increased killing for both strains of bacteria (p < 0.001). 5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one was identified as having broad spectrum activity and consequently represents an excellent candidate for the development of novel surfaces for the prevention of biomedical device infections.     

Development of a multispecies periodontal biofilm model within a stirred bioreactor

Abstract

The objective of this work was to develop a subgingival biofilm model using a stirred bioreactor. Discs of bovine teeth were adapted to a stirred bioreactor filled with a culture medium containing bacterial species associated with periodontal health or disease. After anaerobic incubation, the biofilms growing on the substratum surfaces were collected and analyzed. The mean number of Colony-forming Units (CFUs) varied, but with no difference between 3 and 7?days of biofilm formation (p?>?0.05). Scanning Electron Microscopy (SEM) analysis showed a uniform biofilm layer covering the cement layer of the root surface containing bacteria with diverse morphology. In checkerboard DNA-DNA hybridization, bacterial species were identified in both biofilms. In conclusion, a subgingival biofilm model was developed using a stirred bioreactor, allowing the in vitro reproduction of complex microbial communities. This is an advanced model that may be useful to mimic complex clinical periodontal biofilms.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Potential mechanisms for the effects of tea extracts on the attachment,biofilm formation and cell size of Streptococcus mutans

Tea can inhibit the attachment of Streptococcus mutans to surfaces and subsequent biofilm formation. Five commercial tea extracts were screened for their ability to inhibit attachment and biofilm formation by two strains of S. mutans on glass and hydroxyapatite surfaces. The mechanisms of these effects were investigated using scanning electron microscopy (SEM) and phytochemical screening. The results indicated that extracts of oolong tea most effectively inhibited attachment and extracts of pu-erh tea most effectively inhibited biofilm formation. SEM images showed that the S. mutans cells treated with extracts of oolong tea, or grown in medium containing extracts of pu-erh tea, were coated with tea components and were larger with more rounded shapes. The coatings on the cells consisted of flavonoids, tannins and indolic compounds. The ratio of tannins to simple phenolics in each of the coating samples was ～3:1. This study suggests potential mechanisms by which tea components may inhibit the attachment and subsequent biofilm formation of S. mutans on tooth surfaces, such as modification of cell surface properties and blocking of the activity of proteins and the structures used by the bacteria to interact with surfaces.     

Bacterial composition of biofilms formed on dairy-processing equipment

Abstract

Polymicrobial biofilms often form on the surfaces of food-processing machinery, causing equipment damage and posing a contamination risk for the foods processed by the system. The composition of the microbial communities that make up these biofilms is largely unknown, especially in the dairy industry. To address this deficit, we investigated the bacterial composition of biofilms that form on the surfaces of equipment during dairy processing using Illumina MiSeq sequencing and culture-dependent methods. Illumina sequencing identified eight phyla, comprising six classes, ten orders, fifteen families, eighteen genera, and eighteen species. In contrast, only eight species were isolated from the same samples using the culture-based method. To determine the ability of the identified bacteria to form biofilms, biofilm formation analysis via crystal violet staining was performed. Five of the eight culturable species, Acinetobacter baumannii, Acinetobacter junii, Enterococcus faecalis, Corynebacterium callunae, and Stenotrophomonas maltophilia, were able to form biofilms. Since most of the identified bacteria are potential food-borne or opportunistic pathogens, this study provides guidance for quality control of products produced in dairy processing facilities.     

Identification of critical residues in Gap3 of <Emphasis Type="Italic">Streptococcus parasanguinis</Emphasis> involved in Fap1 glycosylation,fimbrial formation and <Emphasis Type="Italic">in vitro</Emphasis>adhesion

Background  

Streptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation.     

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.     

Multiple drug resistance and strength of attachment to surfaces in Pseudomonas aeruginosa isolates

Aims: To investigate the presence of a relationship between the strength of attachment of Pseudomonas aeruginosa to stainless steel surfaces and their observed multiple drug resistance. Methods and Results: Multiple drug resistance of clinical and environmental isolates of Ps. aeruginosa was evaluated using disc diffusion method. The blot succession technique was used to quantify the strength of attachment of Ps. aeruginosa isolates. Different multiple drug–resistant Ps. aeruginosa isolates exhibited variable attachment strength. Although the highest multiple drug–resistant clinical isolate was shown to have the least attachment strength among clinical isolates, a weak correlation was found between attachment strength and multiple resistance among our investigated Ps. aeruginosa isolates. Conclusions: There is a weak correlation between multiple drug resistance and strength of attachment to stainless steel surfaces. Significance and Impact of the Study: Even low‐resistant Ps. aeruginosa could have the potential of attaching firmly to surfaces and forming biofilm.     

Prevention of Candida albicans Biofilm Formation by Covalently Bound Dimethylaminoethylmethacrylate and Polyethylenimine

Candida albicans biofilms are a major cause of voice prosthesis deterioration in laryngectomized patients. The aim of this study was to produce a surface capable of inhibiting C. albicans biofilm formation. Dimethylaminoethylmethacrylate (DMAEMA) and polyethylenimine (PEI) moieties were covalently bound to the surface of polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA) and subsequently quaternized. Physicochemical characterization of the grafted surfaces was carried out and their effect on C. albicans cell numbers was assessed using a modified Robbins device to grow the biofilms. Covalently bound quaternized polyDMAEMA (polyDMAEMAq) and PEI (PEIq) inhibited biofilm growth, with reductions up to 92%. Our approach may show promise for future application in medical devices such as catheters and prostheses.     

Conditioning film formation and its influence on the initial adhesion and biofilm formation by a cyanobacterium on photobioreactor materials

Abstract

Although cyanobacteria are a common group of microorganisms well-suited to utilization in photobioreactors (PBRs), studies of cyanobacteria fouling and its prevention are scarce. Using a cyanobacterium, Anabaena sp. PCC 7120, which had been genetically modified to enhance linalool production, the formation of conditioning films and the effects of these on the physico-chemical surface properties of various PBR materials during initial adhesion and biofilm formation were investigated. The adhesion assay revealed that the overall attachment of Anabaena was substratum dependent and no correlation between the hydrophobicity/roughness of clean material and cell attachment was found. Surface hydrophilicity/hydrophobicity of all the materials changed within 12?h due to formation of conditioning films. ATR-FTIR spectroscopy revealed that the fractional change in protein deposition between 12 to 96?h was consistent with Anabaena cell attachment but polysaccharide deposition was material specific and did not correlate with cell attachment on the PBR materials. Also, the delay in conditioning film proteins on PVC and PTFE indicated that components other than proteins may be responsible for the decrease in contact angles on these surfaces within 12?h. This indicates the important role of the chemical nature of adsorbed conditioning films in determining the initial attachment of Anabaena to PBR materials. The lower rate of attachment of Anabaena on the hydrophilic surfaces (glass and PMMA) between 72?h to 96?h (regime 3) showed that these surfaces could potentially have low fouling characteristics at extended time scales and should be considered for further research.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

Biofilm formation by haloarchaea

A fluorescence‐based live‐cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P‐value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet‐like, multi‐layered biofilms containing micro‐ and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t‐ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757^T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754^T and Hfx. volcanii DSM 3757^T, cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754^T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.