Associative and Entanglement Contributions to the Solution Rheology of a Bacterial Polysaccharide

We report the viscosity of semidilute solutions of a bacterially synthesized polysaccharidea partially deacetylated poly-<i>N</i>-acetylglucosamineas measured by microrheology. This polymer, commonly called polysaccharide intercellular adhesin (PIA), is synthesized by <i>Staphylococcal</i> strains; it is a principal component of the biofilms of these bacteria. We show that the concentration-dependent viscosity of PIA at a pH in which it is associated can be predicted using the Heo–Larson equation for entangled polymers [J. Rheol. 2005, 49 (5), 1117−1128], if the molecular parameters of the equation are measured in its associated state. This agreement is consistent with PIA adopting a concentration-dependent scaling of the viscosity that is dominated by entanglements and intermolecular associations, as described in the theory of Rubinstein and Semenov [Macromolecules 2001, 34 (4), 1058−1068]. The zero-shear specific viscosity, η_sp, measured in the concentration range, <i>c</i>_PIA = 0.1–13 wt %, scales as η_sp ∼ <i>c</i>_PIA^1.27±0.15 up to an entanglement concentration, <i>c</i>_e = 3.2 wt %, after which η_sp ∼ <i>c</i>_PIA^4.25±0.30. In the presence of urea, a known disruptor of associations, these scaling shifts to η_sp ∼ <i>c</i>_PIA^1.02±0.2 and η_sp ∼ <i>c</i>_PIA^2.57±0.6, respectively; no shift in <i>c</i>_e is observed. The urea effect is consistent with an associative contribution to viscosity in the aqueous solution case. The invariance of <i>c</i>_e suggests that the rheology of this polymer–solvent system also includes an entanglement contribution. With independent estimates of the PIA weight-average molar mass, <i>M</i>_w, entanglement molecular weight, <i>M</i>_e, hydrodynamic radius, <i>R</i>_H, and excluded volume, ν, we use the Heo–Larson equation to predict η_sp as a function of <i>c</i>_PIA. With the use of parameters from the associated stateparticularly the hydrodynamic radiuswe find good agreement between the model and data for aqueous PIA solutions. This study offers a means to predict the rheology of associating polysaccharides using correlations for nonassociating polymers adjusted with minimal <i>a priori</i> data from their associated state

Role of Environmental and Antibiotic Stress on Staphylococcus epidermidis Biofilm Microstructure

Cellular clustering and separation of Staphylococcus epidermidis surface adherent biofilms were found to depend significantly on both antibiotic and environmental stress present during growth under steady flow. Image analysis techniques common to colloidal science were applied to image volumes acquired with high-resolution confocal laser scanning microscopy to extract spatial positions of individual bacteria in volumes of size ∼30 × 30 × 15 μm³. The local number density, cluster distribution, and radial distribution function were determined at each condition by analyzing the statistics of the bacterial spatial positions. Environmental stressors of high osmotic pressure (776 mM NaCl) and sublethal antibiotic dose (1.9 μg/mL vancomycin) decreased the average bacterial local number density 10-fold. Device-associated bacterial biofilms are frequently exposed to these environmental and antibiotic stressors while undergoing flow in the bloodstream. Characteristic density phenotypes associated with low, medium, and high local number densities were identified in unstressed S. epidermidis biofilms, while stressed biofilms contained medium- and low-density phenotypes. All biofilms exhibited clustering at length scales commensurate with cell division (∼1.0 μm). However, density phenotypes differed in cellular connectivity at the scale of ∼6 μm. On this scale, nearly all cells in the high- and medium-density phenotypes were connected into a single cluster with a structure characteristic of a densely packed disordered fluid. However, in the low-density phenotype, the number of clusters was greater, equal to 4% of the total number of cells, and structures were fractal in nature with <i>d</i>_f =1.7 ± 0.1. The work advances the understanding of biofilm growth, informs the development of predictive models of transport and mechanical properties of biofilms, and provides a method for quantifying the kinetics of bacterial surface colonization as well as biofilm fracture and fragmentation

A Cationic Amphiphilic Random Copolymer with pH-Responsive Activity against Methicillin-Resistant <i>Staphylococcus aureus</i>

<div><p>In this report, we demonstrate the pH-dependent, <i>in vitro</i> antimicrobial activity of a cationic, amphiphilic random copolymer against clinical isolates of drug-resistant <i>Staphylococcus aureus</i>. The polymer was developed toward a long-term goal of potential utility in the treatment of skin infections. The proposed mechanism of action of the polymer is through selectively binding to bacterial membranes and subsequent disruption of the membrane structure/integrity, ultimately resulting in bacterial cell death. The polymer showed bactericidal activity against clinical isolates of methicillin-resistant or vancomycin-intermediate <i>S</i>. <i>aureus</i>. The polymer was effective in killing <i>S</i>. <i>aureus</i> at neutral pH, but inactive under acidic conditions (pH 5.5). The polymer did not exhibit any significant hemolytic activity against human red blood cells or display cytotoxicity to human dermal fibroblasts over a range of pH values (5.5–7.4). These results indicate that the polymer activity was selective against bacteria over human cells. Using this polymer, we propose a new potential strategy for treatment of skin infections using the pH-sensitive antimicrobial polymer agent that would selectively target infections at pH-neutral wound sites, but not the acidic, healthy skin.</p></div

Hemolytic activity of PE₃₁ against RBCs.

<p>Reported values are mean and standard deviation of three replicates in duplicate samples.</p

Effect of media pH and acids on <i>S</i>. <i>aureus</i> growth.

<p>24-hour change in bacterial density of <i>S</i>.<i>aureus</i> grown at pH 5.5, 6.5, and 7.4 in MH broth. The pH of MH broth was adjusted by the acids indicated. The initial bacterial suspension contained <i>S</i>. <i>aureus</i> with 2×10⁶ colony-forming units (CFUs) per ml presented by a broken line. Results shown are the mean and standard deviation of three independent experiments in duplicate samples per condition.</p

Susceptibility of MRSA to vancomycin, mupirocin, and PE₃₁.

<p>Susceptibility of MRSA to vancomycin, mupirocin, and PE₃₁.</p

Relationship between pH, bacterial zeta potential, and minimum inhibitory concentration of PE₃₁.

<p>Data reflect multiple replicate zeta-potential measurements and MIC measurements across ten clinical blood isolates of methicillin-resistant <i>S</i>. <i>aureus</i>.</p

Characterization of a reverse-phase perfluorocarbon emulsion for the pulmonary delivery of tobramycin.

<p>BACKGROUND: Aerosolized delivery of antibiotics is hindered by poor penetration within distal and plugged airways. Antibacterial perfluorocarbon ventilation (APV) is a proposed solution in which the lungs are partially or totally filled with perfluorocarbon (PFC) containing emulsified antibiotics. The purpose of this study was to evaluate emulsion stability and rheological, antibacterial, and pharmacokinetic characteristics.</p> <p>METHODS: This study examined emulsion aqueous droplet diameter and number density over 24 hr and emulsion and neat PFC viscosity and surface tension. Additionally, Pseudomonas aeruginosa biofilm growth was measured after 2-hr exposure to emulsion with variable aqueous volume percentages (0.25, 1, and 2.5%) and aqueous tobramycin concentrations (Ca=0.4, 4, and 40 mg/mL). Lastly, the time course of serum and pulmonary tobramycin concentrations was evaluated following APV and conventional aerosolized delivery of tobramycin in rats.</p> <p>RESULTS: The initial aqueous droplet diameter averaged 1.9±0.2 μm with little change over time. Initial aqueous droplet number density averaged 3.5±1.7×10(9) droplets/mL with a significant (p</p> <p>CONCLUSIONS: The emulsion is bactericidal, retains the rheology necessary for pulmonary delivery, is sufficiently stable for this application, and results in increased pulmonary retention of the antibiotic.</p