Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood.

BACKGROUND: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-¹H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. METHODS: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. RESULTS: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at -80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. CONCLUSIONS: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

Making Improvements in the ED: Does ED Busyness Affect Time to Antibiotics in Febrile Pediatric Oncology Patients Presenting to the Emergency Department?

OBJECTIVES: Febrile neutropenic pediatric patients are at heightened risk for serious bacterial infections, and rapid antibiotic administration (in <60 minutes) improves survival. Our objectives were to reduce the time-to-antibiotic (TTA) administration and to evaluate the effect of overall emergency department (ED) busyness on TTA. METHODS: This study was a quality improvement initiative with retrospective chart review to reduce TTA in febrile children with underlying diagnosis of cancer or hematologic immunodeficiency who visited the pediatric ED. A multidisciplinary clinical practice guideline (CPG) was implemented to improve TTA. The CPG's main focus was delivery of antibiotics before availability of laboratory data. We collected data on TTA during baseline and intervention periods. Concurrent patient arrivals to the ED per hour served as a proxy of busyness. Time to antibiotic was compared with the number of concurrent arrivals per hour. Analyses included scatter plot and regression analysis. RESULTS: There were 253 visits from October 1, 2010 to March 30, 2012. Median TTA administration dropped from 207 to 89 minutes (P < 0.001). Eight months after completing all intervention periods, the median had dropped again to 44 minutes with 70% of patients receiving antibiotics within 60 minutes of ED arrival. There was no correlation between concurrent patient arrivals and TTA administration during the historical or intervention periods. CONCLUSIONS: Implementation of a CPG and process improvements significantly reduced median TTA administration. Total patient arrivals per hour as a proxy of ED crowding did not affect TTA administration. Our data suggest that positive improvements in clinical care can be successful despite fluctuations in ED patient volume.

Effect of Antimicrobial and Physical Treatments on Growth of Multispecies Staphylococcal Biofilms.

The prevalence and structure of Staphylococcus aureus and Staphylococcus epidermidis within multispecies biofilms were found to depend sensitively on physical environment and antibiotic dosage. Although these species commonly infect similar sites, such as orthopedic implants, little is known about their behavior in multispecies communities, particularly in response to treatment. This research establishes that S. aureus is much more prevalent than S. epidermidis when simultaneously seeded and grown under unstressed conditions (pH 7, 37°C) in both laboratory and clinical strains. In multispecies communities, S. epidermidis is capable of growing a more confluent biofilm when the addition of S. aureus is delayed 4 to 6 h during 18 h of growth. Different vancomycin dosages generate various behaviors: S. epidermidis is more prevalent at a dose of 1.0 µg/ml vancomycin, but reduced growth of both species occurs at 1.9 µg/ml vancomycin. This variability is consistent with the different MICs of S. aureus and S. epidermidis Growth at higher temperature (45°C) results in an environment where S. aureus forms porous biofilms. This porosity allows S. epidermidis to colonize more of the surface, resulting in detectable S. epidermidis biomass. Variations in pH result in increased prevalence of S. epidermidis at low pH (pH 5 and 6), while S. aureus remains dominant at high pH (pH 8 and 9). This work establishes the structural variability of multispecies staphylococcal biofilms as they undergo physical and antimicrobial treatments. It provides a basis for understanding the structure of these communities at infection sites and how treatments disrupt their multispecies behaviors.IMPORTANCEStaphylococcus aureus and Staphylococcus epidermidis are two species of bacteria that are commonly responsible for biofilm infections on medical devices. Biofilms are structured communities of bacteria surrounded by polysaccharides, proteins, and DNA; bacteria are more resistant to antimicrobials as part of a biofilm than as individual cells. This work investigates the structure and prevalence of these two organisms when grown together in multispecies biofilms and shows shifts in the behavior of the polymicrobial community when grown in various concentrations of vancomycin (an antibiotic commonly used to treat staphylococcal infections), in a high-temperature environment (a condition previously shown to lead to cell disruption and death), and at low and high pH (a change that has been previously shown to soften the mechanical properties of staphylococcal biofilms). These shifts in community structure demonstrate the effect such treatments may have on multispecies staphylococcal infections.

A Cationic Amphiphilic Random Copolymer with pH-Responsive Activity against Methicillin-Resistant Staphylococcus aureus.

In this report, we demonstrate the pH-dependent, in vitro antimicrobial activity of a cationic, amphiphilic random copolymer against clinical isolates of drug-resistant Staphylococcus aureus. 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 S. aureus. The polymer was effective in killing S. aureus 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.

Implantable Device-Related Infection.

Over half of the nearly two million healthcare-associated infections can be attributed to indwelling medical devices. In this review, we highlight the difficulty in diagnosing implantable device-related infection and how this leads to a likely underestimate of the prevalence. We then provide a length-scale conceptualization of device-related infection pathogenesis. Within this conceptualization we focus specifically on biofilm formation and the role of host immune and coagulation systems. Using this framework, we describe how current and developing preventative strategies target specific processes along the entire length-scale. In light of the significant time horizon for the development and translation of new preventative technologies, we also emphasize the need for parallel development of in situ treatment strategies. Specific examples of both preventative and treatment strategies and how they align with the length-scale conceptualization are described.

Zinc oxide nanoparticle suspensions and layer-by-layer coatings inhibit staphylococcal growth.

Despite a decade of engineering and process improvements, bacterial infection remains the primary threat to implanted medical devices. Zinc oxide nanoparticles (ZnO-NPs) have demonstrated antimicrobial properties. Their microbial selectivity, stability, ease of production, and low cost make them attractive alternatives to silver NPs or antimicrobial peptides. Here we sought to (1) determine the relative efficacy of ZnO-NPs on planktonic growth of medically relevant pathogens; (2) establish the role of bacterial surface chemistry on ZnO-NP effectiveness; (3) evaluate NP shape as a factor in the dose-response; and (4) evaluate layer-by-layer (LBL) ZnO-NP surface coatings on biofilm growth. ZnO-NPs inhibited bacterial growth in a shape-dependent manner not previously seen or predicted. Pyramid shaped particles were the most effective and contrary to previous work, larger particles were more effective than smaller particles. Differential susceptibility of pathogens may be related to their surface hydrophobicity. LBL ZnO-NO coatings reduced staphylococcal biofilm burden by >95%. From the Clinical Editor: The use of medical implants is widespread. However, bacterial colonization remains a major concern. In this article, the authors investigated the use of zinc oxide nanoparticles (ZnO-NPs) to prevent bacterial infection. They showed in their experiments that ZnO-NPs significantly inhibited bacterial growth. This work may present a new alternative in using ZnO-NPs in medical devices.

Hemodialysis Catheter Heat Transfer for Biofilm Prevention and Treatment.

Central line-associated bloodstream infections (CLABSIs) are not easily treated, and many catheters (e.g., hemodialysis catheters) are not easily replaced. Biofilms (the source of infection) on catheter surfaces are notoriously difficult to eradicate. We have recently demonstrated that modest elevations of temperature lead to increased staphylococcal susceptibility to vancomycin and significantly soften the biofilm matrix. In this study, using a combination of microbiological, computational, and experimental studies, we demonstrate the efficacy, feasibility, and safety of using heat as an adjuvant treatment for infected hemodialysis catheters. Specifically, we show that treating with heat in the presence of antibiotics led to additive killing of Staphylococcus epidermidis with similar trends seen for Staphylococcus aureus and Klebsiella pneumoniae. The magnitude of temperature elevation required is relatively modest (45-50°C) and similar to that used as an adjuvant to traditional cancer therapy. Using a custom-designed benchtop model of a hemodialysis catheter, positioned with tip in the human vena cava as well as computational fluid dynamic simulations, we demonstrate that these temperature elevations are likely achievable in situ with minimal increased in overall blood temperature.

Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly.

We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism.

Whole Blood Reveals More Metabolic Detail of the Human Metabolome than Serum as Measured by 1H-NMR Spectroscopy: Implications for Sepsis Metabolomics.

Serum is a common sample of convenience for metabolomics studies. Its processing time can be lengthy and may result in the loss of metabolites including those of red blood cells (RBCs). Unlike serum, whole blood (WB) is quickly processed, minimizing the influence of variable hemolysis while including RBC metabolites. To determine differences between serum and WB metabolomes, both sample types, collected from healthy volunteers, were assayed by H-NMR (proton nuclear magnetic resonance) spectroscopy. A total of 34 and 50 aqueous metabolites were quantified from serum and WB, respectively. Free hemoglobin (Hgb) levels in serum were measured, and the correlation between Hgb and metabolite concentrations was determined. Most metabolites detected in serum were at higher concentrations in WB with the exception of acetoacetate and propylene glycol. The 18 unique metabolites of WB included adenosine, AMP, ADP, and ATP, which are associated with RBC metabolism. The use of serum results in the underrepresentation of a number of metabolic pathways including branched-chain amino acid degradation and glycolysis and gluconeogenesis. The range of free Hgb in serum was 0.03 to 0.01 g/dL, and eight metabolites were associated (P ≤ 0.05) with free Hgb. The range of free Hgb in serum samples from 18 sepsis patients was 0.02 to 0.46 g/dL. Whole blood and serum have unique aqueous metabolite profiles, but the use of serum may introduce potential pathway bias. Use of WB for metabolomics may be particularly important for studies in diseases such as sepsis in which RBC metabolism is altered, and mechanical and sepsis-induced hemolysis contributes to variance in the metabolome.

Thermal Augmentation of Vancomycin Against Staphylococcal Biofilms.

Given the increasing evidence of safe application of elevated temperature in other clinical contexts, we consider the potential for supplemental hyperthermia to augment the effects of vancomycin against staphylococci, a major source of postoperative and posttraumatic sepsis. Laboratory reference strains and libraries of clinical blood isolates of Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus, both as planktonic cells and as established biofilms, were assessed for thermosensitivity and increased susceptibility to vancomycin in the setting of thermal treatment. In addition to viability measures, patterns of stress gene expression were assessed with quantitative polymerase chain reaction, and structural changes were measured using quantitative transmission electron microscopy. Laboratory strains of both species had reduced growth and biofilm viability at 45°C, a temperature commonly used in other domains such as adjuvant treatments of malignancy. Blood isolates of S. epidermidis were consistent in this regard as well, but significant between-isolate variability in thermosensitivity was seen in blood isolates of S. aureus. Expression profiling and ultrastructural measurements confirmed that elevated temperature was a substantial stressor with or without vancomycin treatment. Our findings suggest that temperature elevations shown to be tolerated in humans in other settings hold the potential to be used as an adjuvant to antibiotic therapy against staphylococcal biofilms.

Effects of temperature on the morphological, polymeric, and mechanical properties of Staphylococcus epidermidis bacterial biofilms.

Changes in temperature were found to affect the morphology, cell viability, and mechanical properties of Staphylococcus epidermidis bacterial biofilms. S. epidermidis biofilms are commonly associated with hospital-acquired medical device infections. We observed the effect of heat treatment on three physical properties of the biofilms: the bacterial cell morphology and viability, the polymeric properties of the extracellular polymeric substance (EPS), and the rheological properties of the bulk biofilm. After application of a 1 h heat treatment at 45 °C, cell reproduction had ceased, and at 60 °C, cell viability was significantly reduced. Size exclusion chromatography was used to fractionate the extracellular polymeric substance (EPS) based on size. Chemical analysis of each fraction showed that the relative concentrations of the polysaccharide, protein, and DNA components of the EPS were unchanged by the heat treatment at 45 and 60 °C. The results suggest that the EPS molecular constituents are not significantly degraded by the temperature treatment. However, some aggregation on the scale of 100 nm was found by dynamic light scattering at 60 °C. Finally, relative to control biofilms maintained at 37 °C, we observed an order of magnitude reduction in the biofilm yield stress after 60 °C temperature treatment. No such difference was found for treatment at 45 °C. From these results, we conclude that the yield stress of bacterial biofilms is temperature-sensitive and that this sensitivity is correlated with cell viability. The observed significant decrease in yield stress with temperature suggests a means to weaken the mechanical integrity of S. epidermidis biofilms with applications in areas such as the treatment of biofilm-infected medical devices.

Sticky surface: sphere-sphere adhesion dynamics.

We present a multi-scale model to study the attachment of spherical particles with a rigid core, coated with binding ligands and suspended in the surrounding, quiescent fluid medium. This class of fluid-immersed adhesion is widespread in many natural and engineering settings, particularly in microbial surface adhesion. Our theory highlights how the micro-scale binding kinetics of these ligands, as well as the attractive/repulsive surface potential in an ionic medium affects the eventual macro-scale size distribution of the particle aggregates (flocs). The bridge between the micro-macro model is made via an aggregation kernel. Results suggest that the presence of elastic ligands on the particle surface lead to the formation of larger floc aggregates via efficient inter-floc collisions (i.e. non-zero sticking probability, g). Strong electrolytic composition of the surrounding fluid favours large floc formation as well. The kernel for the Brownian diffusion for hard spheres is recovered in the limit of perfect binding effectiveness (g→1) and in a neutral solution with no dissolved salts.

Elasticity of microscale volumes of viscoelastic soft matter by cavitation rheometry.

Measurement of the elastic modulus of soft, viscoelastic liquids with cavitation rheometry is demonstrated for specimens as small as 1 µl by application of elasticity theory and experiments on semi-dilute polymer solutions. Cavitation rheometry is the extraction of the elastic modulus of a material, E, by measuring the pressure necessary to create a cavity within it [J. A. Zimberlin, N. Sanabria-DeLong, G. N. Tew, and A. J. Crosby, Soft Matter 3, 763-767 (2007)]. This paper extends cavitation rheometry in three ways. First, we show that viscoelastic samples can be approximated with the neo-Hookean model provided that the time scale of the cavity formation is measured. Second, we extend the cavitation rheometry method to accommodate cases in which the sample size is no longer large relative to the cavity dimension. Finally, we implement cavitation rheometry to show that the theory accurately measures the elastic modulus of viscoelastic samples with volumes ranging from 4 ml to as low as 1 µl.

Multilocus Sequence Typing for Interpreting Blood Isolates of Staphylococcus epidermidis.

Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is also a common contaminant of blood cultures and, as a result, there is frequently uncertainty as to its diagnostic significance when recovered in the clinical laboratory. One molecular strategy that might be of value in clarifying the interpretation of S. epidermidis identified in blood culture is multilocus sequence typing. Here, we examined 100 isolates of this species (50 blood isolates representing true bacteremia, 25 likely contaminant isolates, and 25 skin isolates) and the ability of sequence typing to differentiate them. Three machine learning algorithms (classification regression tree, support vector machine, and nearest neighbor) were employed. Genetic variability was substantial between isolates, with 44 sequence types found in 100 isolates. Sequence types 2 and 5 were most commonly identified. However, among the classification algorithms we employed, none were effective, with CART and SVM both yielding only 73% diagnostic accuracy and nearest neighbor analysis yielding only 53% accuracy. Our data mirror previous studies examining the presence or absence of pathogenic genes in that the overlap between truly significant organisms and contaminants appears to prevent the use of MLST in the clarification of blood cultures recovering S. epidermidis.

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

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. 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. 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<0.01) decrease over time. Emulsion and PFC viscosity were not significantly different, averaging 1.22±0.03×10(-3) Pa·sec. The surface tensions of PFC and emulsion were 15.0±0.1×10(-3) and 14.6±0.6×10(-3) N/m, respectively, and the aqueous interfacial tensions were 46.7±0.3×10(-3) and 26.9±11.0×10(-3) N/m (p<0.01), respectively. Biofilm growth decreased markedly with increasing Ca and, to a lesser extent, aqueous volume percentage. Tobramycin delivered via APV yielded 2.5 and 10 times larger pulmonary concentrations at 1 and 4 hr post delivery, respectively, and significantly (p<0.05) lower serum concentrations compared with aerosolized delivery. 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.

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 µm(3). 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 d(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.

Molar mass, entanglement, and associations of the biofilm polysaccharide of Staphylococcus epidermidis.

Biofilms are microbial communities that are characterized by the presence of a viscoelastic extracellular polymeric substance (EPS). Studies have shown that polysaccharides, along with proteins and DNA, are a major constituent of the EPS and play a dominant role in mediating its microstructure and rheological properties. Here, we investigate the possibility of entanglements and associative complexes in solutions of extracellular polysaccharide intercellular adhesin (PIA) extracted from Staphylococcus epidermidis biofilms. We report that the weight average molar mass and radius of gyration of PIA isolates are 2.01×10(5)±1200 g/mol and 29.2±1.2 nm, respectively. The coil overlap concentration, c*, was thus determined to be (32±4)×10(-4) g/mL. Measurements of the in situ concentration of PIA (cPIA,biofilm) was found to be (10±2)×10(-4) g/mL.Thus, cPIA,biofilm

Complement c5a generation by staphylococcal biofilms.

Biofilms production is a central feature of nosocomial infection of catheters and other medical devices used in resuscitation and critical care. However, the very effective biofilm forming pathogen Staphylococcus epidermidis often produces a modest host inflammatory response and few of the signs and symptoms associated with more virulent pathogens. To examine the impact of bacterial biofilm formation on provocation of an innate immune response, we studied the elaboration of the major complement anaphylatoxin C5a by human serum upon contact with S. epidermidis biofilms. Wild-type S. epidermidis and mutants of sarA (a regulatory protein that promotes synthesis of the biofilm-forming polysaccharide intercellular adhesin [PIA]) and icaB (responsible for postexport processing of PIA) were studied. C5a release, as a function of exposed biofilm surface area, was on the order of 1 fmol · cm · s and was dependent on the presence of PIA. Experimental results were used to inform a physiologically based pharmacokinetic model of C5a release by an infected central venous catheter, one of S. epidermidis' primary means of causing human disease. These simulations revealed that the magnitude of C5a release on a superior vena cava catheter completely covered with S. epidermidis would be lower than necessary to alert circulating leukocytes. Combined, the experimental and computational results are highly consistent with clinical observations in which the clinical signs of central line-associated bloodstream infection are often muted in association with this important pathogen.