The construction and properties of an integrable plasmid for genomic analysis in Staphylococcus aureus

An improved system for transforming protoplasts of Staphylococcus aureus with plasmid DNA was developed to facilitate the construction of an integrable plasmid. Protoplast transformants were recovered at higher frequencies when the protoplasts were warmed to 56 C immediately prior to the addition of cesium chloride-purified plasmid DNAs. More transformants were recovered when the protoplast-DNA mixture was plated on R agar than on DM3 medium, and when the selective agent was incorporated directly in the medium rather than supplied in an agar overlay. More transformants were recovered when the protoplast-DNA mixture was incubated at 30 C for 3.5 h before plating than when this mixture was plated immediately. The protocol developed in this study yielded higher transformation frequencies than similar procedures for protoplast transformation of S. aureus;The use of a restriction-deficient recipient strain and an improved protocol for protoplast transformation facilitated the isolation of a recombinant plasmid, designated pPQ126, directly in S. aureus. pPQ126 is a novel, temperature-sensitive integrable plasmid 13.6 kilobase pairs in length. Plasmid pPQ126 contains genes encoding for resistance to erythromycin and chloramphenicol (from plasmid pTV1(,ts)) and resistance to gentamicin (from transposon Tn4001). Plasmid pPQ126 is established as an autonomous replicon when introduced into an appropriate recipient strain at the permissive temperature (30 C). Integration of pPQ126 is then directed into homologous target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 C (non-permissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of a chromosomal target. It was also observed that pPQ126 excises from the chromosome under permissive conditions, and excision often generates derivatives of this plasmid that contain additional fragment(s) of DNA

Effective Design Features for the Management of Behavioral Health Patients In General Emergency Departments of Hospitals

This study investigated nursing staff members perspectives of their existing Emergency Department (ED) and their ability to care appropriately for behavioral health patients within the environment. The study involved three rural hospitals in eastern Texas that may not always have the proper resources to care effectively for this vulnerable patient population. The researcher administered a paper-based survey utilizing a Likert-scale response system to nursing staff across all facilities and received participation from 56 respondents. Survey questions were designed to investigate the current ED environment and identify design features available to assist with caring for behavioral health patients. Data gathered revealed staff members’ preference for enhanced security within the ED in addition to designated treatment area(s) to help better manage the behavioral health population treated at their facilities

Microbial shelf life of chub-packaged ground beef from four large U.S. processing plants

Ten pound chubs of coarsely ground beef of two different lean:fat specifications (73:27 and 81:19) were stored at three temperatures (34, 38 or 45 ÌŠF) to monitor the effects of storage temperature on microbial condition of the product. Ground beef from four U.S. plants was tested (2 trials each), and microbial analyses were conducted on storage days 0, 6, 10, 14, and 18 using seven different media to estimate counts of total aerobic and anaerobic, lactic acid bacteria (LAB), and Gram-negative bacteria. Bacterial counts for a given culture medium were similar among plants and meat types. At day 10, total mi crobial counts from chubs stored at 38 or 45 ÌŠF were approximately 8 log10 CFU/g, whereas total counts from chubs stored at 34 ÌŠF were approximately 4.5 log 10 CFU/g (4 log=10,000, CFU is colony forming units). Regardless of storage temperature and meat type, LAB predominated. Growth of gram-negative enteric bacteria was delayed in chubs stored at 34 ÌŠF throughout the 18 day study, whereas counts increased in chubs stored at 38 or 45 ÌŠ F

Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6 s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log10 BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91 ± 0.05 log10 BA spores/mL of milk and the 1.4- μm membrane removed 4.50 ± 0.35 log10 BA spores/ mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log10 spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log10 BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 °C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the protein for arginine and to elucidate structural changes associated with arginine binding. The protein binds arginine with a dissociation constant of 20 mM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima. 2. Molecular organization and structural stability

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscope observations with specific secondary structural elements. The data determined the presence of a buried ẞ-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding bacterial binding protein family members and their potential biotechnological applications