Mechanical deformation of elastomer medical devices can enable microbial surface colonization.

Elastomers such as silicone are common in medical devices (catheters, prosthetic implants, endoscopes), but they remain prone to microbial colonization and biofilm infections. For the first time, our work shows that rates of microbial surface attachment to polydimethylsiloxane (PDMS) silicone can be significantly affected by mechanical deformation. For a section of bent commercial catheter tubing, bacteria (P. aeruginosa) show a strong preference for the 'convex' side compared to the 'concave' side, by a factor of 4.2. Further testing of cast PDMS materials in bending only showed a significant difference for samples that were manually wiped (damaged) beforehand (1.75 × 104 and 6.02 × 103 cells/mm2 on the convex and concave sides, respectively). We demonstrate that surface microcracks in elastomers are opened under tensile stress (convex bending) to become 'activated' as sites for microbial colonization. This work demonstrates that the high elastic limit of elastomers enables these microcracks to reversibly open and close, as 'dynamic defects'. Commercial catheters have relatively high surface roughness inherent to manufacturing, but we show that even manual wiping of newly-cast PDMS is sufficient to generate surface microcracks. We consider the implication for medical devices that feature sustained, surgical, or cyclic deformation, in which localized tensile conditions may expose these surface defects to opportunistic microbes. As a result, our work showcases serious potential problems in the widespread usage and development of elastomers in medical devices.

Long-Term Reduction of Bacterial Adhesion on Polyurethane by an Ultra-Thin Surface Modifier.

Indwelling urinary catheters are employed widely to relieve urinary retention in patients. A common side effect of the use of these catheters is the formation of urinary tract infections (UTIs), which can lead not only to severe medical complications, but even to death. A number of approaches have been used to attempt reduction in the rate of UTI development in catheterized patients, which include the application of antibiotics and modification of the device surface by coatings. Many of these coatings have not seen use on catheters in medical settings due to either the high cost of their implementation, their long-term stability, or their safety. In previous work, it has been established that the simple, stable, and easily applicable sterilization surface coating 2-(3-trichlorosilylpropyloxy)-ethyl hydroxide (MEG-OH) can be applied to polyurethane plastic, where it greatly reduces microbial fouling from a variety of species for a 1-day time period. In the present work, we establish that this coating is able to remain stable and provide a similarly large reduction in fouling against Escherichia coli and Staphylococcus aureus for time periods in an excess of 30 days. This non-specific coating functioned against both Gram-positive and Gram-negative bacteria, providing a log 1.1 to log 1.9 reduction, depending on the species and day. This stability and continued efficacy greatly suggest that MEG-OH may be capable of providing a solution to the UTI issue which occurs with urinary catheters.

Antioxidant and antimicrobial activities and UPLC-ESI-MS/MS polyphenolic profile of sweet orange peel extracts.

With growing consumer awareness, exploitation of renewable resources is cost-effective and environment friendly. This work examines the potential of citrus peels as natural antioxidants and antimicrobials for food preservation. Extraction yield, total soluble phenols and flavonoids of various citrus peels (sweet orange, lemon, tangerine and grapefruit) were optimized by varying the solvent type. While the highest extract yield (~16 â€‹g/100g) was obtained from the sweet orange peels in methanol, extraction with ethanol maximized the concentration of total phenols and flavonoids (~80 â€‹mg catechol equivalents/100 â€‹g dry weight). In addition, sweet orange peel extract showed the highest DPPH, ABTS and hydroxyl radical scavenging values. UPLC-ESI-MS/MS analysis of aqueous and ethanolic extracts of sweet orange peels revealed more than 40 polyphenolic compounds including phenolic acids and flavonoids, some of which have not been previously reported. The predominant polyphenols were narirutin, naringin, hesperetin-7-O-rutinoside naringenin, quinic acid, hesperetin, datiscetin-3-O-rutinoside and sakuranetin. The incorporation of sweet orange peel extract into two vegetable oils enhanced their oxidative stability. In addition, all citrus peel extracts possessed high antimicrobial activity against several food-borne pathogens, and the activity was highest for the sweet orange peel extract. Overall results suggested the great potential of sweet orange peels as natural antioxidant and antimicrobials, which can be efficiently extracted using a simple and low-cost method, for enhancing the storage stability and safety of vegetable oils.

Reduction of microbial adhesion on polyurethane by a sub-nanometer covalently-attached surface modifier.

Indwelling urinary catheters are a common medical device used to relieve urinary retention. Many patients who undergo urinary catheterization develop urinary tract infections (UTIs), which can lead to severe medical complications and high cost of subsequent treatment. Recent years have seen a number of attempts at reducing the rate of UTIs in catheterized patients via catheter surface modifications. In this work, a low cost, robust anti-thrombogenic, and sterilizable anti-fouling layer based on a covalently-bound monoethylene glycol hydroxide (MEG-OH) was attached to polyurethane, a polymeric material commonly used to fabricate catheters. Modified polyurethane tubing was compared to bare tubing after exposure to a wide spectrum of pathogens including Gram-negative bacteria (Pesudomonas aeruginosa, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus) and a fungus (Candida albicans). It has been demonstrated that the MEG-OH monolayer was able to significantly reduce the amount of adhesion of pathogens present on the material surface, with between 85 and 96 % reduction after 24 h of exposure. Additionally, similar reductions in surface fouling were observed following autoclave sterilization, long term storage of samples in air, and longer exposure up to 3 days.

Preventing Pseudomonas aeruginosa Biofilms on Indwelling Catheters by Surface-Bound Enzymes.

Implanted medical devices such as central venous catheters are highly susceptible to microbial colonization and biofilm formation and are a major risk factor for nosocomial infections. The opportunistic pathogen Pseudomonas aeruginosa uses exopolysaccharides, such as Psl, for both initial surface attachment and biofilm formation. We have previously shown that chemically immobilizing the Psl-specific glycoside hydrolase, PslGh, to a material surface can inhibit P. aeruginosa biofilm formation. Herein, we show that PslGh can be uniformly immobilized on the lumen surface of medical-grade, commercial polyethylene, polyurethane, and polydimethylsiloxane (silicone) catheter tubing. We confirmed that the surface-bound PslGh was uniformly distributed along the catheter length and remained active even after storage for 30 days at 4 °C. P. aeruginosa colonization and biofilm formation under dynamic flow culture conditions in vitro showed a 3-log reduction in the number of bacteria during the first 11 days, and a 2-log reduction by day 14 for PslGh-modified PE-100 catheters, compared to untreated catheter controls. In an in vivo rat infection model, PslGh-modified PE-100 catheters showed a ∼1.5-log reduction in the colonization of the clinical P. aeruginosa ATCC 27853 strain after 24 h. These results demonstrate the robust ability of surface-bound glycoside hydrolase enzymes to inhibit biofilm formation and their potential to reduce rates of device-associated infections.

Lubrication dynamics of swollen silicones to limit long term fouling and microbial biofilms.

Bacterial contamination and biofilm formation on medical devices remain a costly and serious healthcare problem. Silicone (polydimethylsiloxane, PDMS) elastomers are common biomaterials but are susceptible to bacterial surface contamination and biofilm growth. 'Self-lubricated' PDMS elastomers (iPDMS) have the potential to greatly reduce rates of cell attachment, biofilm formation and infection. Cross-linked PDMS elastomers immersed in PDMS oil swell to an equilibrium concentration to form a swollen network, and then form a surface liquid layer through syneresis. Herein we have measured the swelling and syneresis kinetics as a function of time, viscosity (1.5 to 10 cSt), and cross-linking density to optimize the surface lubricant layer formation, and resistance to biofouling. The lubricant layer thickness was measured in situ (optical profilometry and AFM) for flat and micro-textured surfaces, as a function of time and swelling ratio, to be in a range from 0.1 to 1 µm, and continuously increases with time. We show this continuous generation is likely due to a gradual, dynamic re-structuring of the elastomer network. Long term antifouling properties of (10 cSt) iPDMS were tested for Pseudomonas aeruginosa growth in a flow culture bioreactor, and after 30 d showed a 103 to 104 reduction of bacterial cell density for iPDMS compared to conventional PDMS elastomers. This long term performance and non-specific activity makes them highly suitable for biomedical devices, such as urinary catheters.

Isolation and characterization of a novel lutein-producing marine microalga using high throughput screening.

A novel highly selective lutein-producing marine microalga was isolated and identified using high throughput screening (HTS). Phylogenetic analysis based on 18S rRNA gene sequence revealed it belongs to a microalgae class (Trebouxiophyceae), and is closely related to Auxenochlorella spp. The novel strain was designated as Auxenochlorella sp. strain LEU27. Based on HPLC-DAD and HPLC-MS analyses, strain LEU27 produced a total of 1203.9 ±â€¯98 µg g-1 dry cells of carotenoids, including a remarkable amount (996.6 ±â€¯98 µg g-1 dry cells) of pure lutein under heterotrophic growth conditions. This is the first report that describes a natural, highly selective lutein-producing marine microalga isolated by HTS approach.

Rapid and Selective Screening Method for Isolation and Identification of Carotenoid-Producing Bacteria.

Carotenoids are naturally occurring yellow to red pigments with many biological activities including antioxidant, anticancer, anti-inflammatory, membrane stabilizers, and precursors for vitamin A. These biological activities are linked with many health benefits (e.g., anticarcinogenic activity, prevention of chronic diseases, etc.), which grew the interest of several industrial sectors especially in food, feed, nutraceuticals, cosmetics, and pharmaceutical industries. The production of natural carotenoids from microbial sources such as bacteria can help meet the growing global market of carotenoids estimated at $1.5 billion in 2014 and is expected to reach 1.8 billion in 2019. This chapter demonstrates, step-by-step, the development of a rapid and selective screening method for isolation and identification of carotenoid-producing microorganisms and their carotenoid analysis. This method involves three main procedures: UV treatment, sequencing analysis of 16S rRNA genes, and carotenoids analysis using rapid and effective HPLC-diode array-MS methods.

Purification and Identification of Astaxanthin and Its Novel Derivative Produced by Radio-tolerant Sphingomonas astaxanthinifaciens.

The red diketocarotenoid, astaxanthin, exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster, which may potentially protect against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue. These numerous health benefits and consumer interest in natural products have therefore increased the market demand of astaxanthin as a nutraceutical and medicinal ingredient in food, aquaculture feed, and pharmaceutical industries. Consequently, many research efforts have been made to discover novel microbial sources with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diode array-MS methods for carotenoids analysis, we isolated a novel astaxanthin-producing bacterium (strain TDMA-17T) that belongs to the family Sphingomonadaceae (Asker et al., FEMS Microbiol Lett 273:140-148, 2007).In this chapter, we provide a comprehensive description of the methods used for the analysis and identification of carotenoids produced by strain TDMA-17T. We will also describe the methods of isolation and identification for a novel bacterial carotenoid (an astaxanthin derivative), a major carotenoid that is produced by the novel strain. Finally, the identification methods of the novel strain will be summarized.

Screening, Isolation, and Identification of Zeaxanthin-Producing Bacteria.

Zeaxanthin is a yellow xanthophyll, dihydroxy-carotenoid, that is naturally found in some of the green, orange, and yellow vegetables and fruits and has a powerful antioxidant activity. Epidemiological evidences suggest that increasing the consumption of zeaxanthin in the diet is associated with a lower risk of age-related macular degeneration (ARMD) and cataracts, two of the leading causes of blindness in the world. Zeaxanthin is a promising nutraceutical/colorant with many applications in feed, food, and pharmaceutical industries. Currently, the commercial production of zeaxanthin is dependent on synthetic routes with limitation in production from biological sources. However, the biotechnological production of natural zeaxanthin is favored due to its safety, potential large-scale production and consumers' preference for natural additives. In this chapter, we describe a rapid screening method based on 16S rRNA gene sequencing and effective HPLC with diode array detector/MS methods for the isolation and identification of zeaxanthin-producing bacteria and their carotenoid analysis.

Food-Safe Modification of Stainless Steel Food-Processing Surfaces to Reduce Bacterial Biofilms.

Biofilm formation on stainless steel (SS) surfaces of food-processing plants, leading to food-borne illness outbreaks, is enabled by the attachment and confinement of pathogens within microscale cavities of surface roughness (grooves, scratches). We report foodsafe oil-based slippery coatings (FOSCs) for food-processing surfaces that suppress bacterial adherence and biofilm formation by trapping residual oil lubricant within these surface cavities to block microbial growth. SS surfaces were chemically functionalized with alkylphosphonic acid to preferentially wet a layer of food-grade oil. FOSCs reduced the effective surface roughness, the adhesion of organic food residue, and bacteria. FOSCs significantly reduced Pseudomonas aeruginosa biofilm formation on standard roughness SS-316 by 5 log CFU cm-2, and by 3 log CFU cm-2 for mirror-finished SS. FOSCs also enhanced surface cleanability, which we measured by bacterial counts after conventional detergent cleaning. Importantly, both SS grades maintained their antibiofilm activity after the erosion of the oil layer by surface wear with glass beads, which suggests that there is a residual volume of oil that remains to block surface cavity defects. These results indicate the potential of such low-cost, scalable approaches to enhance the cleanability of SS food-processing surfaces and improve food safety by reducing biofilm growth.

High throughput screening and profiling of high-value carotenoids from a wide diversity of bacteria in surface seawater.

Carotenoids are valuable natural colorants that exhibit numerous health promoting properties, and thus are widely used in food, feeds, pharmaceutical and nutraceuticals industries. In this study, we isolated and identified novel microbial sources that produced high-value carotenoids using high throughput screening (HTS). A total of 701 pigmented microbial strains library including marine bacteria and red yeast was constructed. Carotenoids profiling using HPLC-DAD-MS methods showed 88 marine bacterial strains with potential for the production of high-value carotenoids including astaxanthin (28 strains), zeaxanthin (21 strains), lutein (1 strains) and canthaxanthin (2 strains). A comprehensive 16S rRNA gene based phylogenetic analysis revealed that these strains can be classified into 30 species belonging to five bacterial classes (Flavobacteriia, α-Proteobacteria, γ-Proteobacteria, Actinobacteria and Bacilli). Importantly, we discovered novel producers of zeaxanthin and lutein, and a high diversity in both carotenoids and producing microbial strains, which are promising and highly selective biotechnological sources for high-value carotenoids.

Non-eluting, surface-bound enzymes disrupt surface attachment of bacteria by continuous biofilm polysaccharide degradation.

Bacterial colonization and biofilm formation on surfaces are typically mediated by the deposition of exopolysaccharides and conditioning protein layers. Pseudomonas aeruginosa is a nosocomial opportunistic pathogen that utilizes strain-specific exopolysaccharides such as Psl, Pel or alginate for both initial surface attachment and biofilm formation. To generate surfaces that resist P. aeruginosa colonization, we covalently bound a Psl-specific glycoside hydrolase (PslGh) to several, chemically-distinct surfaces using amine functionalization (APTMS) and glutaraldehyde (GDA) linking. In situ quartz crystal microbalance (QCM) experiments and fluorescence microscopy demonstrated a complete lack of Psl adsorption on the PslGh-bound surfaces. Covalently-bound PslGh was also found to significantly reduce P. aeruginosa surface attachment and biofilm formation over extended growth periods (8 days). The PslGh surfaces showed a ∼99.9% (∼3-log) reduction in surface associated bacteria compared to control (untreated) surfaces, or those treated with inactive enzyme. This work demonstrates a non-eluting 'bioactive' surface that specifically targets a mechanism of cell adhesion, and that surface-bound glycoside hydrolase can significantly reduce surface colonization of bacteria through local, continuous enzymatic degradation of exopolysaccharide (Psl). These results have significant implications for the surface design of medical devices to keep bacteria in a planktonic state, and therefore susceptible to antibiotics and antimicrobials.

Screening and profiling of natural ketocarotenoids from environmental aquatic bacterial isolates.

Ketocarotenoids are high-value natural pigments. The red diketocarotenoid astaxanthin particularly exhibits an extraordinary antioxidant activity, which raises its market demand for foods and nutraceuticals. We screened for ketocarotenoid-producing bacteria from both marine and freshwater environments. Phylogenetic analysis, based on 16S rRNA gene sequence, revealed 37 potential producers of ketocarotenoids that are related to α-proteobacteria, comprising 32 strains of Brevundimonas and 5 strains of Erythrobacter. Carotenoids analysis by HPLC-DAD and HPLC-MS revealed two groups; astaxanthin-producers (28 Brevundimonas strains) and adonixanthin-producers (Five Brevundimonas and 5 Erythrobacter strains). Strain FrW-Asx16 exhibited the highest carotenoid production (1060 µg g-1 dry cells with 16.6% astaxanthin). Strain FrW-Asx-5 producing 946.1 µg g-1 dry cells carotenoid exhibited the highest astaxanthin content (∼46%). The most intriguing result is the potential of producing natural colorants from freshwater bacterial isolates, and with high productivity and selectivity, suggesting a great promise for their application in food.

Evidence of coexisting microemulsion droplets in oil-in-water emulsions revealed by 2D DOSY 1H NMR.

Optimizing the macroscopic properties, shelf-life and stability of emulsion products requires a better understanding of the microstructural characteristics such as the type (nano, micro and macro) and the relative distribution of components (i.e., oil and surfactant) within the emulsion droplets. We used Diffusion-Ordered NMR Spectroscopy (DOSY NMR) to evaluate these characteristics in model oil-in-water emulsion containing Tween 80 and medium chain triglycerides (MCT). At low MCT concentrations, the solutions were transparent but from 1 to 5 wt% MCT, they became translucent then opaque. 1 wt% MCT was the upper boundary for the appearance of nanoemulsion phase. From the decays of the chemical shift signals of MCT and Tween 80, the DOSY results clearly demonstrate that the self-diffusion coefficients (D) are dependent on oil concentration. Small microemulsion droplets of almost uniform size (d = 12-22 nm) coexist with two sets of large nanoemulsion (d < 200 nm) and emulsion (d > 200 nm) droplets. The large droplets increase significantly in size with increasing MCT. The most striking result is the clear evidence for the presence of microemulsion droplets of nearly uniform size in the aqueous phase from below to above the nanoemulsion transition concentration at 1 wt% MCT.

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness.

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15-35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.

Isolation and Characterization of a Novel, Highly Selective Astaxanthin-Producing Marine Bacterium.

A high-throughput screening approach for astaxanthin-producing bacteria led to the discovery of a novel, highly selective astaxanthin-producing marine bacterium (strain N-5). Phylogenetic analysis based on partial 16S rRNA gene and phenotypic metabolic testing indicated it belongs to the genus Brevundimonas. Therefore, it was designated as Brevundimonas sp. strain N-5. To identify and quantify carotenoids produced by strain N-5, HPLC-DAD and HPLC-MS methods were used. The culture conditions including media, shaking, and time had significant effects on cell growth and carotenoids production including astaxanthin. The total carotenoids were ∼601.2 µg g-1 dry cells including a remarkable amount (364.6 µg g-1 dry cells) of optically pure astaxanthin (3S, 3'S) isomer, with high selectivity (∼60.6%) under medium aeration conditions. Notably, increasing the culture aeration enhanced astaxanthin production up to 85% of total carotenoids. This is the first report that describes a natural, highly selective astaxanthin-producing marine bacterium.