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.

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.

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.

Impact of hourly emergency department patient volume on blood culture contamination and diagnostic yield.

Emergency departments (EDs) are an important diagnostic site for outpatients with potentially serious infections. EDs frequently experience high patient volumes, and crowding has been shown to negatively impact the delivery of early care for serious infections, such as pneumonia. Here, we hypothesized that other important factors in the early care of infectious diseases, the rate of blood culture contamination and the accurate detection of pathogens, would be sensitive to ED operational stress, as proper collection requires fastidious attention to technique and timing. We related all blood samples collected over 1 year and their rates of recovery of likely contaminants and pathogens to the number of patients being cared for in the ED at the time of sample collection. Likely pathogens and contaminants were classified through combined microbiological and manual chart review criteria. Zero-inflated Poisson regression was used to relate crowding to culture results. Blood samples were obtained from 7,586 patients over 82,521 adult and pediatric patient visits. The unadjusted rates of recovering a likely pathogen or a likely contaminant were 8.0% and 3.7%, respectively. Periods of increased crowding (3rd and 4th quartiles of hourly occupancy) were significantly associated (P < 0.01) with increased rates of contamination (relative risk, 1.23 compared to the least busy quartile). Collecting samples for culture during busy times was also associated with a reduced likelihood of recovering a likely pathogen (relative risk, 0.93 compared to the least busy quartile). ED crowding was associated with degraded performance of blood cultures, both increasing the rate of contamination and decreasing the diagnostic yield.

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

In situ rheology of Staphylococcus epidermidis bacterial biofilms.

We developed a method to grow Staphylococcus epidermidis bacterial biofilms and characterize their rheological properties in situ in a continuously fed bioreactor incorporated into a parallel plate rheometer. The temperature and shear rates of growth modeled bloodstream conditions, a common site of S. epidermidis infection. We measured the linear elastic (G') and viscous moduli (G″) of the material using small-amplitude oscillatory rheology and the yield stress using non-linear creep rheology. We found that the elastic and viscous moduli of the S. epidermidis biofilm were 11 ± 3 Pa and 1.9 ± 0.5 Pa at a frequency of 1 Hz (6.283 rad per s) and that the yield stress was approximately 20 Pa. We modeled the linear creep response of the biofilm using a Jeffreys model and found that S. epidermidis has a characteristic relaxation time of approximately 750 seconds and a linear creep viscosity of 3000 Pa s. The effects on the linear viscoelastic moduli of environmental stressors, such as NaCl concentration and extremes of temperature, were also studied. We found a non-monotonic relationship between moduli and NaCl concentrations, with the stiffest material properties found at human physiological concentrations (135 mM). Temperature dependent rheology showed hysteresis in the moduli when heated and cooled between 5 °C and 60 °C. Through these experiments, we demonstrated that biofilms are rheologically complex materials that can be characterized by a combination of low modulus (~10 Pa), long relaxation time (~103 seconds), and a finite yield stress (20 Pa). This suggests that biofilms should be viewed as soft viscoelastic solids whose properties are determined in part by local environmental conditions. The in situ growth method introduced here can be adapted to a wide range of biofilm systems and applied over a broad spectrum of rheological and environmental conditions because the technique minimizes the risk of irreversible, non-linear deformation of the microbial specimen before analysis.

Generation of C5a in the absence of C3: a new complement activation pathway.

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

Multicellularity and antibiotic resistance in Klebsiella pneumoniae grown under bloodstream-mimicking fluid dynamic conditions.

BACKGROUND: While the importance of fluid dynamical conditions is well recognized in the growth of biofilms, their role during bacteremia is unknown. We examined the impact of physiological fluid shear forces on the development of multicellular aggregates of Klebsiella pneumoniae. METHODS: Wild-type and O-antigen or capsular mutants of K. pneumoniae were grown as broth culture in a Taylor-Couette flow cell configured to provide continuous shear forces comparable to those encountered in the human arterial circulation (ie, on the order of 1.0 Pa). The size distribution and antibiotic resistance of aggregates formed in this apparatus were determined, as was their ability to persist in the bloodstream of mice following intravenous injection. RESULTS: Unlike growth in shaking flasks, bacteria grown in the test apparatus readily formed aggregates, a phenotype largely absent in capsular mutants and to a lesser degree in O-antigen mutants. Aggregates were found to persist in the bloodstream of mice. Importantly, organisms grown under physiological shear were found to have an antibiotic resistance phenotype intermediate between that of fully planktonic and biofilm states. CONCLUSIONS: When grown under intravascular-magnitude fluid dynamic conditions, K. pneumoniae spontaneously develops into multicellular aggregates that are capable of persisting in the circulation and exhibit increased antibiotic resistance.

Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures.

We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size.

Prospective trial of real-time electronic surveillance to expedite early care of severe sepsis.

STUDY OBJECTIVE: An automated, real-time electronic medical record query and caregiver notification system was developed and examined for its utility in improving sepsis care. We hypothesize that the algorithm will increase the rate and timeliness of sampling of blood lactate and blood cultures, performance of chest radiography, and provision of antibiotics. METHODS: A before-and-after, prospective study with consecutive enrollment examined an algorithm that automatically identified adult patients accumulating 2 or more systemic inflammatory response syndrome (SIRS) criteria and 2 or more blood pressure measurements less than or equal to 90 mm Hg during their emergency department (ED) stay. In phase 1, the system collected information but did not alert caregivers. In phase 2, caregivers were notified by alphanumeric paging and a text entry into the electronic medical record of the patients' potential illness and were provided with specific recommendations. RESULTS: Patients (33,460) were screened during 6 months; 398 patients activated the system, including 184 (46%) appropriately identified as severely septic. The algorithm had a 54% positive predictive value and 99% negative predictive value in detecting severe infection with acute organ dysfunction. The median time for patients to accumulate SIRS and blood pressure criteria was 152 minutes (interquartile range [IQR] 71 to 284 minutes), underscoring the dynamic nature of diagnosing critical illness in the emergency setting and the need for detection algorithms to repeatedly assess patients during their evaluation. After implementation, 2 interventions were performed more frequently, chest radiograph before admission (odds ratio 3.2; 95% confidence interval 1.1 to 9.5) and collection of blood cultures (odds ratio 2.9; 95% confidence interval 1.1 to 7.7). Only blood culture testing was performed significantly faster in the presence of decision support (median time to culture before intervention 86 minutes, IQR 31, 296 minutes; median time to culture after intervention 81 minutes, IQR 37, 245 minutes; P=.032 by Cox proportional hazards modeling). The predominant shortcoming of the strategy was failing to detect severely septic cases before caregivers. CONCLUSION: An automated algorithm for detecting potential sepsis increased the frequency and timeliness of some ED interventions for severe sepsis. Future efforts need to identify patient features present earlier in ED evaluation than SIRS and hypotension.

Diabetes is not associated with increased mortality in emergency department patients with sepsis.

STUDY OBJECTIVE: Despite its high prevalence, the influence of diabetes on outcomes of emergency department (ED) patients with sepsis remains undefined. Our aim is to investigate the association of diabetes and initial glucose level with mortality in patients with suspected infection from the ED. METHODS: Three independent, observational, prospective cohorts from 2 large US tertiary care centers were studied. We included patients admitted to the hospital from the ED with suspected infection. We investigated the association of diabetes and inhospital mortality within each cohort separately and then overall with logistic regression and generalized estimating equations adjusted for age, sex, disease severity, and sepsis syndrome. We also tested for an interaction between diabetes and hyperglycemia/hypoglycemia. RESULTS: A total of 7,754 patients were included. The mortality rate was 4.3% (95% confidence interval [CI] 3.9% to 4.8%) and similar in diabetic and nondiabetic patients (4.1% versus 4.4%; absolute risk difference 0.4%; 95% CI -0.7% to 1.4%). There was no significant association between diabetes and mortality in adjusted analysis (odds ratio [OR] overall 0.85; 95% CI 0.71 to 1.01). Diabetes significantly modified the effect of hyperglycemia and hypoglycemia with mortality; initial glucose levels greater than 200 mg/dL were associated with higher mortality in nondiabetic patients (OR 2.1; 95% CI 1.4 to 3.0) but not in diabetic patients (OR 1.0; 95% CI 0.2 to 4.7), whereas glucose levels less than 100 mg/dL were associated with higher mortality mainly in the diabetic population (OR 2.3; 95% CI 1.6 to 3.3) and to a lesser extent in nondiabetic patients (OR 1.1; 95% CI 1.03 to 1.14). CONCLUSION: We found no evidence for a harmful association of diabetes and mortality in patients across different sepsis severities. High initial glucose levels were associated with adverse outcomes in the nondiabetic population only. Further investigation is warranted to determine the mechanism for these effects.

Short-term functional decline of older adults admitted for suspected sepsis.

INTRODUCTION: We investigated the degree of functional decline and loss of independence among older adults presenting to the emergency department (ED) with serious infection and to estimate 90-day case fatality. METHODS: Consecutive patients 70 years or older (n = 50) presenting to the ED with potentially serious infection were identified using an automated case-identification algorithm. Fifty age- and sex-matched controls were recruited from a registry of community volunteers. Functional and residential statuses were ascertained at hospital admission, discharge, and 90 days. Details regarding patients' comorbidities, acute illness, and diagnostic evaluation were collected along with 90-day survival. RESULTS: Older adults with suspected sepsis had substantial 90-day mortality (32.0%). Baseline functional impairment was more severe among cases than among control subjects, although activities of daily living and instrumental activities of daily living deficits did not predict outcome. Hospital admission was also not uniformly associated with deterioration in either activities of daily living or instrumental activities of daily living performance. Patients admitted from home were at no greater risk of functional decline than were those admitted from care facilities. No enrolled case enjoyed an increase in residential independence after discharge; of the 28 who were independent upon admission, 19 survived to 90 days, and 8 of the 19 required visiting assistance or were in a care facility. CONCLUSIONS: Older adults presenting to the ED with potentially serious infection have significant 90-day mortality. Although functional status does not consistently deteriorate in survivors, there seems to be considerable risk in the short-term for loss of residential independence.

Dynamics of human complement-mediated killing of Klebsiella pneumoniae.

With an in vitro system that used a luminescent strain of Klebsiella pneumoniae to assess bacterial metabolic activity in near-real-time, we investigated the dynamics of complement-mediated attack in healthy individuals and in patients presenting to the emergency department with community-acquired severe sepsis. A novel mathematical/statistical model was developed to simplify light output trajectories over time into two fitted parameters, the rate of complement activation and the delay from activation to the onset of killing. Using Factor B-depleted serum, the alternative pathway was found to be the primary bactericidal effector: In the absence of B, C3 opsonization as measured by flow cytometry did not progress and bacteria proliferated near exponentially. Defects in bacterial killing were easily demonstrable in patients with severe sepsis compared with healthy volunteers. In most patients with sepsis, the rate of activation was higher than in normal subjects but was associated with a prolonged delay between activation and bacterial killing (P < 0.05 for both). Theoretical modeling suggested that this combination of accentuated but delayed function should allow successful bacterial killing but with significantly greater complement activation. The use of luminescent bacteria allowed for the development of a novel and powerful tool for assessing complement immunology for the purposes of mechanistic study and patient evaluation.

Accuracy of non-contact infrared thermometry versus rectal thermometry in young children evaluated in the emergency department for fever.

OBJECTIVE: We evaluated the accuracy of a non-contact infrared thermometer compared with a rectal thermometer. METHODS: Two hundred patients, ages 1 month to 4 years, were included in the study. Each child underwent contemporaneous standard rectal thermometry and mid forehead non-contact infrared thermometry. Clinical features, including chief complaint, recently administered antipyretic agents, and ambient temperature at the time of measurement, were included. ANALYSIS: Linear models were used to compare agreement between the 2 techniques, as well as to determine bias of infrared thermometry at different rectal temperatures. Multivariate linear models were used to evaluate the impact of clinical variables and ambient temperature. RESULTS: A linear relationship between rectal and infrared temperature measurements was observed; however, the coefficient of determination (r(2)) value between was only 0.48 (P < 0.01). Infrared thermometry tended to overestimate the temperature of afebrile children and underestimate the temperature of febrile patients (P < .01). Ambient temperature and child age did not affect the accuracy of the device. CONCLUSION: In this study, non-contact infrared thermometry did not sufficiently agree with rectal thermometer to indicate its routine use.

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.

Lipopolysaccharide O-antigen promotes persistent murine bacteremia.

Bacteremia is a common complication of pneumonia with Klebsiella pneumoniae. In the previous work, we have shown that the lipopolysaccharide (LPS) O-antigen in K. pneumoniae O1:K2 contributes to lethality during pneumonia in part by promoting bacteremia. In the current work, we studied an O-antigen-deficient K. pneumoniae strain to further evaluate this polysaccharide's role in bloodstream infection. Cultured macrophage and murine bacteremia models were studied. In vitro, O-antigen-deficient bacteria, compared with wild-type organisms, were stronger activators of the murine alveolar macrophage cell line MH-S as assessed by nuclear localization of RelA/p65 and by secretion of cytokines and chemokines. O-antigen-deficient Klebsiellae were also more susceptible to killing by murine neutrophils. In vivo, the absence of O-antigen allowed more rapid and complete clearance of bacteria from the bloodstream, liver, and spleen after intravenous injection in mice. Survival was also greater among animals infected with bacteria missing the O-antigen. Gene expression profiling (via reverse transcriptase-polymerase chain reaction of 84 inflammatory mediator complementary DNA) revealed that by 24 h postinfection, the livers and spleens of animals infected with O-antigen-deficient organisms had significantly downregulated cytokine and chemokine expression compared with wild-type infected animals. The O-antigen surface carbohydrate of O1:K2 serotype K. pneumoniae appears to contribute to bacterial virulence by lessening the activation of macrophages, conveying resistance to killing by neutrophils, and by promoting persistent infection in the blood, liver, and spleen after the onset of bacteremia.

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.