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

Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil.

Projeto: 04.10.06.001

From DNA sequence to application: possibilities and complications

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF’s) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

High sensitivity and label-free oligonucleotides detection using photonic bandgap sensing structures biofunctionalized with molecular beacon probes

A label-free sensor, based on the combination of silicon photonic bandgap (PBG) structures with immobilized molecular beacon (MB) probes, is experimentally developed. Complementary target oligonucleotides are specifically recognized through hybridization with the MB probes on the surface of the sensing structure. This combination of PBG sensing structures and MB probes demonstrates an extremely high sensitivity without the need for complex PCR-based amplification or labelling methods

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

Experimental study of the evanescent-wave photonic sensors response in presence of molecular beacon conformational changes

An experimental study of the influence of the conformational change suffered by molecular beacon (MB) probes ‐upon the biorecognition of nucleic acid target oligonucleotides over evanescent wave photonic sensors‐ is reported. To this end, high sensitivity photonic sensors based on silicon photonic bandgap (PBG) structures were used, where the MB probes were immobilized via their 5’ termination. Those MBs incorporate a biotin moiety close to their 3’ termination in order to selectively bind a streptavidin molecule to them. The different photonic sensing responses obtained towards the target oligonucleotide detection, when the streptavidin molecule was bound to the MB probes or not, demonstrate the conformational change suffered by the MB upon hybridization, which promotes the displacement of the streptavidin molecule away from the surface of the photonic sensing structure. Schematic diagram of the PBG sensing structure on which the streptavidin‐labeled MB probes were immobilized