David E. Nivens

Role of Alginate and Its O Acetylation in Formation of Pseudomonas aeruginosa Microcolonies and Biofilms

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm(-1). All strains produced biofilms that absorbed IR radiation near 1,650 cm(-1) (amide I), 1,550 cm(-1) (amide II), 1,240 cm(-1) (P==O stretching, C---O---C stretching, and/or amide III vibrations), 1,100 to 1,000 cm(-1) (C---OH and P---O stretching) 1,450 cm(-1), and 1,400 cm(-1). The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm(-1) (C---OH stretching of alginate) and 1,250 cm(-1) (C---O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 micrometer, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 micrometer in depth. In contrast, FRD1 produced microcolonies that were approximately 40 micrometer in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture.

Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation.

Effect of temperature and moisture on the miscibility of amorphous dispersions of felodipine and poly(vinyl pyrrolidone)

The physical stability of amorphous molecular level solid dispersions will be influenced by the miscibility of the components. The goal of this work was to understand the effects of temperature and relative humidity on the miscibility of a model amorphous solid dispersion. Infrared spectroscopy was used to evaluate drug-polymer hydrogen bonding interactions in amorphous solid dispersions of felodipine and poly(vinyl pyrrolidone) (PVP). Samples were analyzed under stressed conditions: high temperature and high relative humidity. The glass transition temperature (T(g)) of select systems was studied using differential scanning calorimetry (DSC). Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were used to further investigate moisture-induced changes in solid dispersions. Felodipine-PVP solid dispersions showed evidence of adhesive hydrogen bonding interactions at all compositions studied. The drug-polymer intermolecular interactions were weakened and/or less numerous on increasing the temperature, but persisted up to the melting temperature of the drug. Changes in the hydrogen bonding interactions were found to be reversible with changes in temperature. In contrast, the introduction of water into amorphous molecular level solid dispersions at room temperature irreversibly disrupted interactions between the drug and the polymer resulting in amorphous-amorphous phase separation followed by crystallization. DSC, AFM, and TEM results provided further evidence for the occurrence of moisture induced immiscibility. In conclusion, it appears that felodipine-PVP solid dispersions are susceptible to moisture-induced immiscibility when stored at a relative humidity >or=75%. In contrast, the solid dispersions remained miscible on heating.

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of Mucoid Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with cystic fibrosis (CF), leading to biofilm-associated infections. The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1, calcium addition (1.0 and 10 mM as CaCl(2)) resulted in biofilms that were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.

Microbial inactivation properties of a new antimicrobial/ antithrombotic catheter lock solution (citrate/methylene blue/parabens)

Background. Microbial infections are the most serious complications associated with indwelling central venous catheters. A catheter lock solution that is both antibacterial and antithrombotic is needed. The goal of this study was to determine whether a new catheter lock solution containing citrate, methylene blue and parabens has antimicrobial properties against planktonic bacteria and against sessile bacteria within a biofilm. These effects were compared to the antimicrobial properties of heparin at 2500 units/ml. Methods. The tested solution (C/MB/P comprising 7% sodium citrate, 0.05% methylene blue and 0.165% parabens) and individual components were challenged against gram-positive and gram-negative organisms and fungi. Control solutions were heparin with preservatives. Studies included evaluation of eradication of planktonic bacteria and sessile organisms in a biofilm grown on polymeric and glass coupons. Biofilm samples were inspected by scanning electron microscopy, atomic force microscopy and vital stains. Results. The C/MB/P solution, contrary to heparin, kills most tested planktonic microorganisms within 1 h of incubation. All tested organisms have an MIC of 25% or less of the original concentration of a new catheter lock. Bacteria strains did not develop resistance over more than 40 passages of culture suspensions. The C/MB/P solution is able to kill nearly all sessile bacteria in biofilm growth on plastic or glass discs in 1 h. Microscopic methods demonstrated extensive physical elimination of biofilm deposits from treated coupons. In contrast, heparin had a minimal effect on planktonic or biofilm organisms. Conclusions. The new multicomponent lock solution has strong antimicrobial properties against both planktonic and sessile microorganisms. By comparison, heparin with preservative has weak antibacterial properties against planktonic and biofilm bacteria. The tested catheter lock may have usefulness in preventing bacterial colonization of haemodialysis catheters and diminishing the incidence of catheter-related bacteraemia.

Sol-gel derived materials as substrates for neuronal differentiation: effects of surface features and protein conformation

This work demonstrates the ability of sol-gel derived materials to support the differentiation of neuronal cells, and investigates the physiochemical interactions between the surface and extracellular matrix proteins as a mediator of the effects of surface features on differentiation. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study the conformational changes of human serum fibronectin, a critical extracellular cell adhesion protein, after adsorption onto native and poly-L-lysine doped sol-gel derived silica thin films and bulk materials. The global conformation of fibronectin varied dramatically between native and organically modified materials and most interestingly between thin films and bulk materials of the same chemistry. A comparison of the surface topography of thin films and bulk materials by atomic force microscopy reveals that films of native silica have surface features less than the AFM tip size (<25 nm) while bulk materials of the same precursor chemistry have features ranging from 50–100 nm in size. Fibronectin assumed an inactive, globular, solution-like state on the larger feature size bulk gels and an active, fully extended fibrillar-like state on the smaller feature size films. Neither native nor PLL-doped bulk materials could support cell growth or neuronal differentiation of PC12 cells, in stark contrast to the thin films, which supported a robust neuronal phenotype. Morphological analysis and expression levels of the neuronal proteins β-tubulin and neurofilament, in addition to the FRET data, indicate that the effects of surface chemistry on fibronectin conformation, cellular adhesion, and differentiation are dependent upon the surface topography.

Peptide ormosils as cellular substrates

Peptide-functionalized thin films exhibit significant potential for integration into implantable devices and cell-based technologies. A new type of neuroactive peptide-modified silica was developed using sol–gel reaction chemistry to produce thin films from four different peptide silane precursors. Peptide silanes containing binding sequences from laminin (YIGSR and KDI), fibronectin (RGD), and EGF repeats from laminin and tenascin (NID) were produced using standard solid-state FMOC peptide synthesis conditions and the covalent attachment of 3′-(aminopropyl)trimethoxysilane (APTMS), using carbonyldiimadazole (CDI) as a linking molecule. Precursor formation was confirmed with MALDI-MS. Thin films were produced by dip-coating using the peptide precursors combined with hydrolyzed tetramethoxysilane. Atomic force microscopy indicated that the surface topography was not affected by low concentrations of peptide precursor (0.0025 mol%), but higher concentrations of peptide precursor (0.01 mol%) resulted in features that were 50–75 nm in height. The height features observed were consistent in size with previously determined topographical morphology supportive of neuronal cell lines. The surfaces were biologically active and modulated the phenotype of the embryonic carcinoma stem cell line, P19. Combinations of the peptide silanes resulted in altered cell types after retinoic acid treatment. More neurons were observed on RGD/YIGSR and RGD/YIGSR/NID surfaces compared to tetramethoxysilane (TMOS) controls. More supporting cells were observed compared to collagen coated tissue culture plates. In addition, neurites were significantly longer on the peptide ormosils compared to controls. This work demonstrates a novel method for producing biologically active peptide ormosils using peptide-modified precursors.

Interactions Between Chemical Functionality and Nanoscale Surface Topography Impact Fibronectin Conformation and Neuronal Differentiation on Model Sol-gel Silica Substrates

Functional relationships between the biomaterial interface and extracellular matrix (ECM) proteins are intimately involved in cellular adhesion and function. Structural changes of ECM proteins upon adsorption to a surface alter the proteins biological activity by varying the availability of molecular binding sites. Recent work using native and organically modified sol-gel silica as a neuronal biointerface revealed that changes in surface nanotopography of bulk versus thin film materials result in dramatic differences in fibronectin structure, cell survival, and neuronal differentiation. In order to further investigate interactions between chemical functionality and surface topography, we evaluated the global conformation of human fibronectin adsorbed to seven different organically modified silica gels and thin films. Chemical functional groups were introduced into the materials either by altering the starting precursor or by doping with poly-l-lysine or polyethylenimine. Surface topography measurements by atomic force microscopy show that films have surface features less than 25 nm while bulk materials of the same precursor chemistry have features ranging from 50 – 100 nm in size. Fluorescence resonance energy transfer spectroscopy (FRET) revealed a strong interaction between surface topography and chemical functionality. Fibronectin remain globular on all bulk materials regardless of chemical modification. The same changes in precursors or dopant chemistry, however, induced changes in the conformation of fibronectin on the thin films. The differentiation of PC12 cells on the surface indicated a strong impact of the surface features and suggest a possible optimal fibronectin folding state.

Sampling and quantification of biofilms in food processing and other environments.

Abstract: In the food industry, assessment of food contact surfaces is necessary to determine whether equipment is properly cleaned and/or sanitized and whether living problematic microorganisms are present. Existing quantitative detection technologies are limited by the inability to directly detect living cells in sporadically dispersed biofilms on large surface areas. Thus, precise and accurate sampling strategies must be coupled with detection technology. This chapter discusses sampling methods and standard (e.g., plating and ATP-bioluminescence) and emerging (e.g., spectrometry, immunosensor, and nucleic acid-based) quantitative techniques to detect biofilms on food contact surfaces with a survey of function, analytical performance, and limitations.

Effects of Low Dose Gamma-Radiation on Select Wheat Properties

The effects of space-travel-relevant radiation doses (gamma radiation) on structure, function, and antioxidant properties of select wheat cultivars (Triticum aestivum L.) were investigated. Following radiation treatment, proteins were analyzed using SDS-PAGE, mixograph, and atomic force microscopy methods. Starch granule damage and pasting curve functionality were evaluated; lipid oxidation was determined using a thiobarbituric acid reactive substances method; and antioxidant capacity was measured using 2,2-diphenyl-1-picrylhydrazyl analyses. Increasing radiation exposure resulted in significant differences between wheat cultivars. Therefore, low-dose gamma-radiation affects wheat, and differences exist between cultivars, so careful consideration is needed when selecting wheat for use in elevated radiation conditions. The Apogee cultivar is likely a better NASA candidate crop than Perigee; however, Yecora Rojo, Parshall, and/or Yavaros 79 cultivars may be more stable to radiation and provide better food functionality traits. Ultimately, wheat should be protected from radiation exposure during space travel, if possible, to maintain quality.