Antifouling Potential of Lubricious,Micro-engineered,PDMS Elastomers against Zoospores of the Green Fouling Alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5?μm wide pillars or ridges separated by 5, 10, or 20?μm wide channels. Pattern depths were 5 or 1.5?μm. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5?μm wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5?μm, 10?μm and 20?μm. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Development of engineered biofilms on poly- L-lysine patterned surfaces

A technique has been developed to selectively attach bacteria to solid supports using poly-l-lysine. The patterned biofilms were labeled with green fluorescent protein (GFP) or a nucleic acid stain and imaged using both confocal microscopy and GFP stereomicroscopy. E. coli DH10B, E. coli MC1061, and Pseudomonas sp. GJ1 were selectively attached to regions coated with poly-l-lysine but not to uncoated regions. In contrast, E. coli DH5, W3110 and 33456 attached indiscriminately to the coated and uncoated regions of the surface. Those organisms that selectively attached to the poly-l-lysine coated regions formed biofilms twice as thick as the organisms that attached indiscriminately to the surface. This technique can be used for selectively patterning surfaces with genetically engineered microorganisms for biosynthesis of secondary metabolites and biodegradation or for developing a bacterial-based microscale medical diagnostic tool.     

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda

Abstract

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ～38?kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01–5?μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2?×?10⁴ ?2?×?10⁹ CFU/ml, and exhibited a lower detection limit of 2?×?10⁴ CFU/ml.     

Effect of adsorbed fibronectin on the differential adhesion of osteoblast-like cells and Staphylococcus aureus with and without fibronectin-binding proteins

The influence of fibronectin (Fn) coated surfaces patterned with poly(ethylene glycol) microgels having inter-gel spacings between 0.5 and 3.0 μm on the adhesion of Staphylococcus aureus strains with and without Fn-binding proteins and cellular adhesion/spreading was investigated. Quantitative force measurements between a S. aureus cell and a patterned surface showed that the adhesion force between the bacterium and the patterned surface increased substantially after Fn adsorption, regardless of the strain used, but decreased with decreasing inter-gel spacing. In flow-chamber experiments, the Fn-binding strain adhered at a higher rate after Fn adsorption than the strain lacking Fn-binding proteins. In both cases, the adhesion rates decreased with decreasing inter-gel spacing. Osteoblast-like cells could bind to patterned surfaces despite the microgels, and adsorbed Fn substantially amplified this effect. Even under highly non-adhesive conditions associated with closely spaced microgels, adsorbed Fn preserves a window of inter-gel spacing around 1 μm where the adhesion of staphylococcal cells is hindered while cells can still adhere and spread.     

Evaluation of Petrifilm™ EC method for enumeration of E. coli from soil

Aims: To evaluate the suitability of commercially available Petrifilm? EC plates for enumeration of Escherichia coli from soil. Methods and Results: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10², 10³, 10⁵ CFU g^?1 of soil. The efficiency of recovery on Petrifilm? EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m‐FC basal medium supplemented with 3‐bromo‐4‐chloro‐5‐indoyl‐β‐d ‐glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4‐methylumbelliferyl‐β‐d ‐glucuronide (EC‐MUG) broth. Petrifilm? EC and m‐FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between Petrifilm? EC, m‐FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure‐applied field soil samples showed a significant difference (P < 0·05) between the Petrifilm? EC method and the m‐FC method in enumerating E. coli possibly as a result of false positives on m‐FC. Conclusion: The Petrifilm? EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g^?1 soil. Significance and Impact of the Study: The commercially available Petrifilm? EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.     

Impact of Delftia tsuruhatensis and Achromobacter xylosoxidans on Escherichia coli dual-species biofilms treated with antibiotic agents

Recently it was demonstrated that for urinary tract infections species with a lower or unproven pathogenic potential, such as Delftia tsuruhatensis and Achromobacter xylosoxidans, might interact with conventional pathogenic agents such as Escherichia coli. Here, single- and dual-species biofilms of these microorganisms were characterized in terms of microbial composition over time, the average fitness of E. coli, the spatial organization and the biofilm antimicrobial profile. The results revealed a positive impact of these species on the fitness of E. coli and a greater tolerance to the antibiotic agents. In dual-species biofilms exposed to antibiotics, E. coli was able to dominate the microbial consortia in spite of being the most sensitive strain. This is the first study demonstrating the protective effect of less common species over E. coli under adverse conditions imposed by the use of antibiotic agents.     

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84–7.25 CFU cm^?2 at 192?h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.     

Engineered antifouling microtopographies – correlating wettability with cell attachment

Abstract

Bioadhesion and surface wettability are influenced by microscale topography. In the present study, engineered pillars, ridges and biomimetic topography inspired by the skin of fast moving sharks (Sharklet AF?) were replicated in polydimethylsiloxane elastomer. Sessile drop contact angle changes on the surfaces correlated well (R² = 0.89) with Wenzel and Cassie and Baxter's relationships for wettability. Two separate biological responses, i.e. settlement of Ulva linza zoospores and alignment of porcine cardiovascular endothelial cells, were inversely proportional to the width (between 5 and 20 μm) of the engineered channels. Zoospore settlement was reduced by ～85% on the finer (ca 2 μm) and more complex Sharklet AF? topographies. The response of both cell types suggests their responses are governed by the same underlying thermodynamic principles as wettability.     

Fate of genetically modified microorganisms in the corn rhizosphere

The fates of genetically modified (GM)Escherichia coli andPseudomonas putida in the corn rhizosphere were investigated. Under hydrophonic and sterile conditions, both bacteria grew well in the presence of root exudates used as a sole carbon source. The growth patterns of wild types and genetically modified strains ofE. coli andP. putida were similar under the conditions tested.The presence of rhizospheric microorganisms affected the survival pattern ofE. coli. In the presence of corn roots and rhizospheric microorganisms,E. coli numbers increased during the first 3 days but were later drastically reduced, probably as a result of competition with rhizospheric microorganisms for the carbon source. However, in the presence of rhizospheric microorganisms,P. putida survived better thanE. coli in the simulated corn rhizosphere.     

Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1–10?μm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28?days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4?μm PDMS surfaces. Also, field deployments over 28?days showed a significant reduction in biovolume on the 4 and 10?μm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10?μm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.     

A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

Abstract

This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.     

Sequence analysis of the gene encoding H antigen in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from food in Morocco

In order to develop other molecular method useful for typing of motile and non motile Escherichia coli strains, a total of 207 strains of E. coli (133 reference strains, 74 food strains) were characterized by analysis of sequences of their amplified flagellin-encoding (fliC) gene products. The collection of reference strains was used for database building of fliC gene sequences. Application of this identification system to 74 E. coli food isolates revealed a reproducible and clear cut classification with very good correlation to results obtained by HhaI restriction of the amplified flagellin gene. The proposed determination of fliC sequences variations should be helpful for epidemiological studies.     

Using textured PDMS to prevent settlement and enhance release of marine fouling organisms

The antifouling efficacy of a series of 18 textured (0.2–1000 μm) and non-textured (0 μm) polydimethylsiloxane surfaces with the profiles of round- and square-wave linear grating was tested by recording the settlement of fouling organisms in the laboratory and in the field by monitoring the recruitment of a multi-species fouling community. In laboratory assays, the diatoms Nitzschia closterium and Amphora sp. were deterred by all surface topographies regardless of texture type. Settlement of propagules of Ulva sp. was lower on texture sizes less than the propagule size, and settlement of larvae of Saccostrea glomerata and Bugula neritina was lower on texture sizes closest to, but less than, the sizes of larvae. After a six month field trial, all textured surfaces lost their deterrent effect; however, the foul-release capabilities of textures were still present. High initial attachment was correlated with most fouling remaining after removal trials, indicating that fouling organisms recruited in higher numbers to surfaces upon which they attached most strongly.     

Effects of a clay mineral on microbial predation and parasitism ofEscherichia coli

Montmorillonitic clay influences the biological control ofEscherichia coli in aquatic systems, the magnitude of the effects being dependent on the state of the clay and the type of host-antagonist interaction. The interaction ofBdellovibrio andE. coli was partially inhibited by the presence of montmorillonite. Because it is highly motile,Bdellovibrio apparently could penetrate any colloidal clay barrier aroundE. coli if the clay envelope was thin enough. Colloidal clay had little effect on predation ofE. coli by the myxobacteriumPolyangium, and had no effect on the activity of the amoebaVexillifera. Crude clay, on the other hand, resulted in a physical separation of predator and prey, and this completely inhibited theE. coli-Polyangium interaction and slowed the rate of engulfment ofE. coli byVexillifera.The interference of natural biological control by clays may alter the microbial balance favoring survival of fecal microorganisms and resulting in their accumulation in saline sediments. This could constitute a health hazard if these organisms were released by upwelling of bottom waters or were desorbed in estuarine systems by dilution during heavy rains.     

合成生物学方法构建电活性大肠杆菌

电活性微生物具有独特的在细胞内外环境之间传递电子的能力。在对天然电活性微生物电子传递机制充分研究的基础上,通过合成生物学方法异源构建天然电活性微生物电子传递结构基础也可以将遗传背景清晰的非电活性大肠杆菌改造为电活性微生物。构建获得的工程化电活性大肠杆菌可以直接应用于微生物燃料电池和生物传感器等领域,同时也可以作为底盘细胞整合相应的目标产物合成通路实现电能驱动的生物合成。本文以合成生物学方法构建电活性大肠杆菌为主题,详细阐述天然电活性微生物电子传递的机理及结构基础,总结了工程化电活性大肠杆菌的构建策略、成功案例以及应用领域,并对合成生物学方法构建电活性大肠杆菌未来的研究方向进行了展望。     

Hyperhalophilic archaeal biofilms: growth kinetics,structure, and antagonistic interaction in continuous culture

Biofilms by the hyperhalophilic archaea Halorubrum sp. and Halobacterium sp. were analyzed, and for the first time the progression of structural features and the developmental parameters of these sessile populations are described. Optical slicing and digital analysis of sequential micrographs showed that their three dimensional structure was microorganism dependent. Biofilms of Halobacterium sp. developed in clusters that covered about 30% of the supporting surface at the interface level and expanded over about 86?±?4 μm in thickness, while Halorubrum sp. biofilms covered less than 20% of the surface and reached a thickness of 41?±?1 μm. The kinetics of growth was lower in biofilms, with generation times of 27?±?1 and 36?±?2 h for Halobacterium sp. and Halorubrum sp., respectively, as compared to 8.4?±?0.3 and 14?±?1 h in planktonic cultures. Differences between microorganisms were also observed at the cell morphology level. The interaction between the two microorganisms was also evaluated, showing that Halobacterium sp. can outcompete already established Halorubrum sp. biofilms by a mechanism that might include the combined action of tunnelling swimmers and antimicrobial compounds.     

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces

Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli‐expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curli‐expressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0·05), indicating produce surfaces did not affect (P < 0·05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0·05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (S_R) to intact and cut produce surfaces increased with time. Conclusions: Curli‐producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.     

On-chip Escherichia coli culture, purification, and detection of expressed proteins

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST–GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.M. Kim and S. Y. Lee contributed equally to this work.     

Role of nutrient limitation in the competition between uropathogenic strains of Klebsiella pneumoniae and Escherichia coli in mixed biofilms

Klebsiella pneumoniae and Escherichia coli form mixed species biofilms in catheter-associated urinary tract infections. Recently, a detrimental effect of K. pneumoniae over E. coli was observed in mixed species biofilms grown in an artificial urine medium. The mechanism behind this competitive interaction was studied. K. pneumoniae partially outcompeted E. coli in early-stage batch-fed biofilms, whereas both microorganisms co-exist at longer times (K. pneumoniae:E. coli ratio, 55:1), as shown by cell counts and confocal microscopy. E. coli cells were scattered along the K. pneumoniae biofilm. Biofilm supernatants did not appear to contain either antimicrobial or anti-biofilm activities against E. coli. Biofilms grown under continuous flow prevented interspecies competition. K. pneumoniae showed both increased siderophore production and better growth in iron-limited media compared to E. coli. In summary, these results indicate the importance of nutrient (particularly iron) competition in the modulation of the bacterial composition of mixed species biofilms formed by uropathogenic K. pneumoniae and E. coli.