Stockpiling versus Composting: Effectiveness in Reducing Antibiotic-Resistant Bacteria and Resistance Genes in Beef Cattle Manure.

Manure storage methods can affect the concentration and prevalence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in cattle manure prior to land application. The objective of this study was to compare stockpiling and composting with respect to their effectiveness in reducing ARB and ARGs in beef cattle manure in a field-scale study. Field experiments were conducted in different seasons with different bulking agents for composting. For both the winter-spring cycle and the summer-fall cycle, ARB concentrations declined below the limit of quantification rapidly in both composting piles and stockpiles; however, ARB prevalence was significantly greater in the composting piles than in the stockpiles. This was likely due to the introduction of ARB from bulking agents. There was no significant change in ARG concentrations between initial and final concentrations for either manure storage treatment during the winter-spring cycle, but a significant reduction of the ARGs erm(B), tet(O), and tet(Q) over time was observed for both the composting pile and stockpile during the summer-fall cycle. Results from this study suggest that (i) bulking agent may be an important source of ARB and ARGs for composting; (ii) during cold months, the heterogeneity of the temperature profile in composting piles could result in poor ARG reduction; and (iii) during warm months, both stockpiling and composting can be effective in reducing ARG abundance. IMPORTANCE Proper treatment of manure is essential to reduce the spread of antibiotic resistance and protect human health. Stockpiling and composting are two manure storage methods which can reduce antibiotic-resistant bacteria and resistance genes, although few field-scale studies have examined the relative efficiency of each method. This study examined the ability of both methods in both winter-spring and summer-fall cycles, while also accounting for heterogeneity within field-scale manure piles. This study determined that bulking agents used in composting could contribute antibiotic-resistant bacteria and resistance genes. Additionally, seasonal variation could hinder the efficacy of composting in colder months due to heterogeneity in temperature within the pile; however, in warmer months, either method of manure storage could be effective in reducing the spread of antibiotic resistance.

Methods for simultaneous determination of legacy and insensitive munition (IM) constituents in aqueous, soil/sediment, and tissue matrices.

Currently, no standard method exists for analyzing insensitive munition (IM) compounds in environmental matrices, with or without concurrent legacy munition compounds, resulting in potentially inaccurate determinations. The primary objective of this work was to develop new methods of extraction, pre-concentration, and analytical separation/quantitation of 17 legacy munition compounds along with several additional IM compounds, IM breakdown products, and other munition compounds that are not currently included in U. S. Environmental Protection Agency (EPA) Method 8330B. The eight additional compounds included were nitroguanidine, 3-nitro-1,2,4-triazol-5-one, picric acid, 2,4-dinitroanisole, 2,4-dinitrophenol, 2-nitrophenol, 4-nitrophenol, and new surrogate ortho-nitrobenzoic acid (o-NBA). Analytical methods were developed to enable sensitive, simultaneous detection and quantitation of the 24 IM and legacy compounds, including two orthogonal high-performance liquid chromatography (HPLC) column separations with either ultraviolet (UV) or mass spectrometric (MS) detection. Procedures were developed for simultaneous extraction of all 24 analytes and two surrogates (1,2-dinitrobenzene, 1,2-DNB; o-NBA) from high- and low-level aqueous matrices and solid matrices, using acidification, solid phase extraction (SPE), or solvent extraction (SE), respectively. For low-level aqueous samples extracted by SPE, all compounds were recovered within current Department of Defense Quality Systems Manual (DoD QSM) Ver5.3 accepted limits for aqueous samples analyzed by EPA Method 8330B (57-135%), except NQ, which was consistently recovered at approximately 50%. Likewise, all compounds were recovered from six geographically/geochemically unique soil types within current QSM accepted limits for solid samples analyzed by EPA Method 8330B (64-135%). Further, the majority of compounds were recovered from four tissue types within current limits for solids, with generally low recovery only for Tetryl (from 4 to 62%). A preparatory chromatographic interference removal procedure was adapted for tissue extracts, as various analytical interferences were observed for all studied tissue types.

Binding Capacity and Selectivity of Functionalized and Un-functionalized Carbon Nanotubes for Development of Copper-Detecting Printable Sensor.

Carbon nanotubes (CNTs) have unique properties which can be modified through surface functionalization. The ability of several functionalized and un-functionalized CNTs to bind copper was investigated as a first step toward developing a printable CNT-based sensor to detect copper in aqueous systems. Binding capacity and specificity were shown to vary by functionalization and vendor. CNTs from two vendors were tested, and the equilibrium binding data was fitted using two isotherm models. Calculated qmax (mg/g) values indicated one vendor's carboxyl-functionalized CNTs had the greatest binding capacity (94-115 mg/g), while other carboxyl-functionalized CNTs and amine-functionalized CNTs had similar capacities to un-functionalized CNTs (15-30 mg/g). Hydroxyl-functionalized CNTs had the lowest copper binding capacity (7-8 mg/g) of the CNTs tested. Freundlich isotherms showed no obvious trends in binding affinity, but suggested that binding was primarily due to chemisorption. Variations in CNT size, functionalization percentage, and purity could explain, partially, the observed adsorption differences.

Multicolored Protein Nanoparticles: Synthesis, Characterization, and Cell Uptake.

Synthesis, characterization, and applications of strongly fluorescent, multicolored protein nanoparticles (GlowDots) are reported here. Bovine serum albumin was cross-linked under controlled conditions to form nanoparticles, where particle size was controlled from 20 to 100 ± 10 nm by choosing appropriate reaction conditions. The absorption as well as the emission wavelengths were controlled without changing the particle size, unlike quantum dots. Each GlowDot was loaded with up to 214 ± 50 chromophores, and hence, the particles have high molar absorptivities (106 M-1 cm-1) as well as high brightness (105 to 106 M-1 cm-1). A large number of functional groups cover the particle surface and these are further functionalized to enhance cellular uptake. GlowDots that were labeled with fluorescein and functionalized with taurine, for example, were quickly taken up by HeLa, MDA-MB-231, PC3, and L6 myoblast cells, as interrogated by fluorescence imaging studies. GlowDots were biocompatible, size tunable, biodegradable, strongly fluorescent, and stable for months at room temperature, and they may serve as substitutes for quantum dots in a variety of practical applications.

Tylosin sorption to diatomaceous earth described by Langmuir isotherm and Freundlich isotherm models.

Tylosin, an antibiotic used for maintaining livestock health, is a macrolide structurally similar to a number of important, often prescribed human antibiotics. Because of this relationship, tylosin presents a potential threat of antimicrobial resistance from environmental buildup. This work investigated tylosin sorption to natural diatomaceous earth product (DE) and the types of physical interactions responsible for sorption. Most sorption processes were best described by the Langmuir model when compared with Freundlich model. Heat of sorption (ΔH) was 1.14 kJ mol-1 indicating a physisorption process. Change in entropy (ΔS) was 119 J mol-1. Sorption was evaluated from aqueous solution with various H+, KCl and Urea concentrations. In 0.01 M phosphate buffer (PB) pH 6.6, a maximum sorption capacity of 15 mg tylosin per g of DE was achieved. Changing the pH to 2.9 or 11.2 resulted in decreased sorption of tylosin (13 and 10 mg g-1, respectively). Addition of 1 M KCl to 0.01 M PB pH 6.6 decreased sorption of tylosin to DE with the maximum binding capacity of 7 mg g-1. Sorption in 1.0 M urea, 0.01 M phosphate buffer pH 6.6 showed a maximum sorption of 13 mg g-1. Based on these results, the sorption of tylosin appears to be a physisorption process, with charge-charge interactions being the mode of sorption at neutral pH and small contributions from secondary interactions. This information will be useful for developing effective strategies for mitigating tylosin and other antimicrobial's impact on the environment.

Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.