Terri A. Camesano

Micelle formation and CMC of gemini surfactants: a thermodynamic model

Gemini surfactants possess a structure resembling a pair of conventional single chain surfactants covalently connected by a spacer. The spacer can vary in length and chemical structure. In this paper, the aggregation behavior of gemini surfactants is examined on the basis of a free energy model developed by extending our theory for conventional surfactants. Free energy contributions beyond those considered for conventional surfactants arise because of the spacer. They originate from the fact that (a) the spacer shields the hydrophobic core of the aggregate from contact with water, (b) the spacer constrains the distance between the head groups, thus imposing non-uniformity in charge distribution at the aggregate surface, (c) the spacer prevents the two linked tails from having a packing conformation inside the micelle identical to that of analogous unlinked single chain surfactants, and (d) the spacer can be partially buried inside the micelle core provided its length and the molecular interactions allow it. Illustrative calculations for gemini surfactants with tail lengths of 5‐16 carbon atoms and various spacers are discussed here. The spacers considered include the polymethylene chain and a combination of polymethylene chain with NCH3 ,O , S, and aromatic groups. The predicted critical micelle concentration (CMC), micelle aggregation number, sphere-to-rod transition parameter (K) and shape of aggregates are compared with available experimental data. A comparative analysis of the magnitude and shape dependence of the individual contributions to the free energy shows that the nonuniform charge distribution at the aggregate surface and the additional packing constraint on the tails, both originating from the short length of the spacer, play a central role in determining the equilibrium shape of the aggregates.

Nanoscale Investigation of Pathogenic Microbial Adhesion to a Biomaterial

ABSTRACT Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between C. parapsilosis and both unmodified silicone rubber and P. aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 ± 0.25 nN in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 ± 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that C. parapsilosis may adhere to both silicone rubber and P. aeruginosa biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.

Cranberries and Their Bioactive Constituents in Human Health

Recent observational and clinical studies have raised interest in the potential health effects of cranberry consumption, an association that appears to be due to the phytochemical content of this fruit. The profile of cranberry bioactives is distinct from that of other berry fruit, being rich in A-type proanthocyanidins (PACs) in contrast to the B-type PACs present in most other fruit. Basic research has suggested a number of potential mechanisms of action of cranberry bioactives, although further molecular studies are necessary. Human studies on the health effects of cranberry products have focused principally on urinary tract and cardiovascular health, with some attention also directed to oral health and gastrointestinal epithelia. Evidence suggesting that cranberries may decrease the recurrence of urinary tract infections is important because a nutritional approach to this condition could lower the use of antibiotic treatment and the consequent development of resistance to these drugs. There is encouraging, but limited, evidence of a cardioprotective effect of cranberries mediated via actions on antioxidant capacity and lipoprotein profiles. The mixed outcomes from clinical studies with cranberry products could result from interventions testing a variety of products, often uncharacterized in their composition of bioactives, using different doses and regimens, as well as the absence of a biomarker for compliance to the protocol. Daily consumption of a variety of fruit is necessary to achieve a healthy dietary pattern, meet recommendations for micronutrient intake, and promote the intake of a diversity of phytochemicals. Berry fruit, including cranberries, represent a rich source of phenolic bioactives that may contribute to human health.

Blocking and ripening of colloids in porous media and their implications for bacterial transport

A model accounting for the dynamics of colloid deposition in porous media was developed and applied to systems containing similarly charged particles and collectors. Colloid breakthrough and intracolumn retention data confirmed that blocking reduced overall colloidal adhesion to soil. The surface coverage at which blocking occurred varied for the type of colloid, as shown by changes in the clean-bed collision efficiency, a0, and the excluded area parameter, b. Excluded area parameters were relatively high due to unfavorable interactions between particles and collectors, and ranged from 11.5 for one bacterium (Pseudomonas putida KT2442) to 13.7 and 24.1 for carboxylated latex microspheres with differing degrees of charged groups on their surfaces. Differences in b values for the three colloids were correlated with electrophoretic mobility, with the most negatively charged colloid (carboxylated latex; CL microspheres) having the highest b. No correlation between hydrophobicity and a0 or b was found. Besides using colloidal particles capable of blocking, the addition of chemical additives to the soil has been suggested as a means for reducing attachment of colloids to porous media. Dextran addition caused an order-of-magnitude reduction in the overall a (for carboxylated-modified latex; CMLs). This reduction was not attributed to blocking, but to the sorption of dextran to the soil which lowered a0. The filtration-based numerical model used to fit the a0 and b parameters was used to demonstrate that blocking could result in significantly enhanced bacterial transport in field situations.

Adhesion forces between Staphylococcus epidermidis and surfaces bearing self-assembled monolayers in the presence of model proteins

Self-assembled monolayers (SAMs) are being developed into coatings to reduce microbial biofilm formation on biomaterials. To test anti-adhesion properties, SAMs can be easily constructed on gold, and used to represent a coated biomaterial. However, coatings that prevent bacterial adhesion must also resist protein adsorption. We explored the competitive effects of bacteria and protein for adsorption to SAMs, choosing fetal bovine serum (FBS) to represent protein non-specific binding, and fibronectin (FN) to evaluate ligand/receptor binding. Staphylococcus epidermidis were immobilized on an atomic force microscope (AFM) tip and used as a force probe to detect the interaction forces between bacteria and gold-coated SAMs. The SAMs tested were alkanethiol molecules terminating in isophthalic acid (IPA) or isophthalic acid with silver (IAG). While S. epidermidis showed weak interactions with FBS, the bacteria showed strong adhesion with FN, due to ligand/receptor binding. Bacterial retention and viability experiments were correlated with the force measurements. S. epidermidis interacting with IAG SAMs showed a loss of viability, due to the mobility of silver ions. For most substrata, there was a link between high adhesion forces with bacteria and a high percentage of dead cells being retained on that substratum (even in the absence of a specific biocidal effect, such as silver). This may suggest that high adhesion forces can cause stress to the bacteria which contributed to their death. The relationship between highly adhesive SAMs and bacterial inactivation may be useful in future biomaterial design. When evaluating coatings for biomaterials, it is important to consider the interplay between bacteria, proteins, and the coating material.

Atomic force microscopy study of the role of LPS O-antigen on adhesion of E. coli

The O‐antigen is a highly variable component of the lipopolysaccharide (LPS) among Escherichia coli strains and is useful for strain identification and assessing virulence. While the O‐antigen has been chemically well characterized in terms of sugar composition, physical properties such as O‐antigen length of E. coli LPS have not been well studied, even though LPS length is important for determining binding of bacteria to biomolecules and epithelial cells. Atomic force microscopy (AFM) was used to characterize the physicochemical properties of the LPS of eight E. coli strains. Steric repulsion between the AFM tip (silicon nitride) and the E. coli cells was measured and modeled, to determine LPS lengths for three O157 and two O113 E. coli strains, and three control (K12) strains that do not express the O‐antigen. For strains with an O‐antigen, the LPS lengths ranged from 17 ± 10 to 37 ± 9 nm, and LPS length was positively correlated with the force of adhesion (Fadh). Longer lengths of LPS may have allowed for more hydrogen bonding between the O‐antigen and silanol groups of the AFM silicon nitride tip, which controlled the magnitude of Fadh. For control strains, LPS lengths ranged from 3 ± 2 to 5 ± 3 nm, and there was no relationship between LPS length and adhesion force between the bacterium and the silicon nitride tip. In the absence of the O‐antigen, we attributed Fadh to electrostatic interactions with lipids in the bacterial membrane. Copyright

Cranberry changes the physicochemical surface properties of E. coli and adhesion with uroepithelial cells.

Cranberries have been suggested to decrease the attachment of bacteria to uroepithelial cells (UC), thus preventing urinary tract infections, although the mechanisms are not well understood. A thermodynamic approach was used to calculate the Gibbs free energy of adhesion changes (DeltaG(adh)) for bacteria-UC interactions, based on measuring contact angles with three probe liquids. Interfacial tensions and DeltaG(adh) values were calculated for Escherichia coli HB101pDC1 (P-fimbriated) and HB101 (non-fimbriated) exposed to cranberry juice (0-27 wt.%). HB101pDC1 can form strong bonds with the Gal-Gal disaccharide receptor on uroepithelial cells, while HB101-UC interactions are only non-specific. For HB101 interacting with UC, DeltaG(adh) was always negative, suggesting favorable adhesion, and the values were insensitive to cranberry juice concentration. For the HB101pDC1-UC system, DeltaG(adh) became positive at 27wt.% cranberry juice, suggesting that adhesion was unfavorable. Acid-base (AB) interactions dominated the interfacial tensions, compared to Lifshitz-van der Waals (LW) interactions. Exposure to cranberry juice increased the AB component of the interfacial tension of HB101pDC1. LW interactions were small and insensitive to cranberry juice concentration. The number of bacteria attached to UC was quantified in batch adhesion assays and quantitatively correlated with DeltaG(adh). Since the thermodynamic approach should not agree with the experimental results when specific interactions are present, such as HB101pDC-UC ligand-receptor bonds, our results may suggest that cranberry juice disrupts bacterial ligand-UC receptor binding. These results help form the mechanistic explanation of how cranberry products can be used to prevent bacterial attachment to host tissue, and may lead to the development of better therapies based on natural products.

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

Binding, inactivation, and adhesion forces between antimicrobial peptide cecropin P1 and pathogenic E. coli

The antimicrobial peptide cecropin P1 (CP1) exhibits broad spectrum activity against planktonic bacteria, including Escherichia coli (E. coli). However, its activity when attached to a substrate has not been thoroughly studied. We immobilized CP1 to gold or silicon nitride, and studied how the method of attachment of peptide to the surface affected peptide interaction with and killing of the bacteria. Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we characterized non-specific binding between CP1 to silicon nitride and gold, and covalent binding of cysteine-terminated CP1 (CP1-cys) to gold. The density of CP1-cys adsorbed on gold was more than the density of CP1 on silicon nitride, and activity against E. coli also depended on the method of attachment used to anchor the peptide to the surface. Twelve E. coli strains with known lipopolysaccharide (LPS) structures were studied. Bacterial adhesion with CP1 was strongest for E. coli with long O-antigens, as determined by atomic force microscopy (AFM). This may be caused by CP1 interacting with the hydrophilic part of the LPS, while control bacteria or those with short O-antigens had their hydrophobic lipid A region more exposed. Killing of E. coli due to contact with CP1 was dependent on the method by which the peptide was immobilized. Four out of 12 E. coli strains were killed when contacted with CP1-cys bound to gold via a thiol bond, while all 12 strains could be killed when in contact with CP1 on silicon nitride. In summary, both QCM-D adsorption experiments and adhesion forces measured by AFM showed a relationship between bacteria LPS length and binding or interaction with the antimicrobial peptide, but killing of E. coli by the peptide was most strongly dependent on how the peptide was attached to the surface.

Atomic Force Microscopy Study of the Effect of Lipopolysaccharides and Extracellular Polymers on Adhesion of Pseudomonas aeruginosa

The roles of lipopolysaccharides (LPS) and extracellular polymers (ECP) on the adhesion of Pseudomonas aeruginosa PAO1 (expresses the A-band and B-band of O antigen) and AK1401 (expresses the A-band but not the B-band) to silicon were investigated with atomic force microscopy (AFM) and related to biopolymer physical properties. Measurement of macroscopic properties showed that strain AK1401 is more negatively charged and slightly more hydrophobic than strain PAO1 is. Microscopic AFM investigations of individual bacteria showed differences in how the biopolymers interacted with silicon. PAO1 showed larger decay lengths in AFM approach cycles, suggesting that the longer polymers on PAO1 caused greater steric repulsion with the AFM tip. For both bacterial strains, the long-range interactions we observed (hundreds of nanometers) were inconsistent with the small sizes of LPS, suggesting that they were also influenced by ECP, especially polysaccharides. The AFM retraction profiles provide information on the adhesion strength of the biopolymers to silicon (F(adh)). For AK1401, the adhesion forces were only slightly lower (F(adh) = 0.51 nN compared to 0.56 nN for PAO1), but the adhesion events were concentrated over shorter distances. More than 90% of adhesion events for AK1401 were at distances of <600 nm, while >50% of adhesion events for PAO1 were at distances of >600 nm. The sizes of the observed molecules suggest that the adhesion of P. aeruginosa to silicon was controlled by ECP, in addition to LPS. Steric and electrostatic forces each contributed to the interfacial interactions between P. aeruginosa and the silicon surface.