Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

“The Ultimate Decision Is Yours”: Exploring Patients’ Attitudes about the Overuse of Medical Interventions

Previous research has found that American patients strongly believe that more testing and more treatment lead to better outcomes and, to a lesser extent, that newer treatments are more effective. We conducted five focus groups with privately insured, healthy, middle-aged Americans (n = 43) to explore these apparent preferences. Contrary to previous research, an unexpected distinction emerged. Participants placed enormous value on testing and screening, reacting with hostility to guidelines recommending less of either. However, they were suspicious of overmedication. The wariness of pharmaceuticals and enthusiasm for testing and screening both appear to reflect participants’ efforts to take responsibility for their health. But recommendations to test and screen less conflicted with their active, engaged, information-seeking roles. Nonetheless, given patients’ concerns about overuse of pharmaceuticals, we maintain that they can learn to understand the connections between over-testing and over-treatment, and can actively choose to do less. We close with suggestions about how treatment guidelines can better communicate these connections to patients. Our findings cannot necessarily be generalized beyond privately-insured, healthy, middle-aged Americans. But because we found that, among these individuals, attitudes towards pharmaceuticals differ from attitudes towards testing and screening, we maintain that future research should also distinguish among and compare attitudes towards different types of medical interventions.     

Classification of Bacteria and Archaea: Past,present and future

The late 19th century was the beginning of bacterial taxonomy and bacteria were classified on the basis of phenotypic markers. The distinction of prokaryotes and eukaryotes was introduced in the 1960s. Numerical taxonomy improved phenotypic identification but provided little information on the phylogenetic relationships of prokaryotes. Later on, chemotaxonomic and genotypic methods were widely used for a more satisfactory classification. Archaea were first classified as a separate group of prokaryotes in 1977. The current classification of Bacteria and Archaea is based on an operational-based model, the so-called polyphasic approach, comprised of phenotypic, chemotaxonomic and genotypic data, as well as phylogenetic information. The provisional status Candidatus has been established for describing uncultured prokaryotic cells for which their phylogenetic relationship has been determined and their authenticity revealed by in situ probing.     

Fluorescence in situ hybridization for intracellular localization of nifH mRNA

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.     

Die Beeinflussung der Aminosäuresequenz des serinhaltigen Mureins von Staphylococcus epidermidis Stamm 24 durch die Nährbodenzusammensetzung

Zusammenfassung Beim Wachstum von S. epidermidis, Stamm 24, in Hefe-Dextrose-Bouillon weist das Murein folgende Molverhältnisse auf (auf- bzw. abgerundete Zahlen): Mur-GlcNH₂:Ala:Glu:Lys:Gly:Ser=1:1:2,4:1:1:4,2:0,6. Die Glutaminsäure ist amidiert.Durch Isolierung und Identifizierung der Peptide des Partialhydrolysates des Mureins wurde die Aminosäuresequenz bestimmt. Die an die Muraminsäure gebundene Peptiduntereinheit (l-Ala-d-GluNH₂ -l-Lys-d-Ala) stimmt mit der von S. aureus, Copenhagen bzw. S. epidermidis, Stamm 66, überein. Bei knapp einem Drittel der Peptiduntereinheiten ist das C-terminale d-Alanin der Mureinvorstufe nicht abgespalten, so daß diese noch als Pentapeptide vorliegen. Dies konnte aus dem Verhältnis l-Ala/d-Ala (1:1,3), dem Ergebnis der Hydrazinolyse und der Isolierung der Muropeptide nach Spaltung der Zellwände mit Lysozym geschlossen werden.Bei etwa der Hälfte aller aus 5 Glycinresten aufgebauten Interpeptidketten ist ein Glycinrest durch l-Serin ersetzt. Die genaue Position des Serins konnte nicht bestimmt werden. Serin ist sicher nicht direkt an die -Aminogruppe des Lysins gebunden.In selteneren Fällen kann Lysin mit l-Alanin substituiert sein, das N-terminal vorliegt und nicht der Quervernetzung dient.Die Dinitrophenylierung des Mureins ergab, daß in etwa 3,5% der Fälle die Interpeptidketten fehlen und rund ein Drittel der Interpeptidketten nicht quervernetzt ist.Bei Wachstum in einem halbsynthetischen, glycinarmen Medium (Minimal, medium) nimmt der Glycinanteil des Mureins um rund 40% ab, während l-Alanin zunimmt. Es konnte gezeigt werden, daß rund 15% des Mureins ein an die -Amino-gruppe des Lysins gebundenes l-Alanin enthalten, das aber im Unterschied zu S. epidermidis, Stamm 66, nicht mit Glycin substituiert ist, sondern N-terminal bleibt und nicht zur Quervernetzung benützt werden kann. Weiterhin liegen hier rund 35% des Lysins unsubstituiert vor, und nur etwa 50% der Peptiduntereinheiten weisen eine Pentaglycyl-Interpeptidkette auf. Die Quervernetzung des Mureins ist bei den in Minimalmedium gewachsenen Zellen nur zu rund 30% durchgeführt. Bei Zusatz von Glycin zum Minimal-Nährboden wird der Glycingehalt im Murein erhöht, während der extra Alaninanteil praktisch verschwindet. Serinzusatz erhöht nicht nur den Serin-, sondern auch den Glycinanteil. Bei Alaninzusatz dagegen wird der Alaningehalt im Murein etwas erhöht und der Glycingehalt weiter erniedrigt.Die Ergebnisse dieser Untersuchungen wurden mit entsprechenden, vorläufigen Versuchen bei S. epidermidis, Stamm 66 und S. aureus, Stamm Copenhagen, verglichen. Es zeigt sich, daß trotz starker modifikativer Veränderungen der Mureinzusammensetzung eindeutige genetische Unterschiede zwischen diesen drei Stämmen vorliegen.

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The effect of nutrition on the amino acid sequence of the serine containing murein of Staphylococcus epidermis strain 24

Summary The murein (peptidoglycan) of S. epidermidis strain 24 contains Mur GlcNH₂, Ala, Glu, Lys, Gly, Ser at a molar ratio of about 1:1:2.4:1:1:4.2:0.6 when grown in a yeast extract dextrose medium. Glutamic acid occurs as an amide.The amino acid sequence was determined by analysing the oligopeptides from partial acid hydrolysate. The tetrapeptide bound to the muramic acid (l-Ala-d-Glu-NH₂-l-Lys-d-Ala) is identical with those found in S. aureus and S. epidermidis strain 66. About 1/3 of the muropeptides is still present as pentapeptides, since the second d-alanine of the muramyl pentapeptide precursor is not split off. This fact is indicated by the ratio of l-Ala/d-Ala of 1:1.3, the isolation of muropentapeptides from the lysozyme lysates and by the result of the hydrazinolysis.About 50% of the pentaglycine interpeptide chains contain one mole of l-serine. The exact position of l-serine could not be determined. However, it could be shown, that serine is never bound to the -amino group of lysine. In very rare cases, the -amino group of lysine is substituted by l-alanine which remains N-terminal and can not be used for crosslinkages.As shown by dinitrophenylation, about 3.5% of the -amino groups of lysine is free and about 50% of the interpeptide chains are not cross-linked.If the organism is grown in a glycine deficient minimal medium, the glycine content of the murein drops by 40%, while l-alanine increases. Here, about 15% of the -amino groups of lysine is substituted by l-alanine, which again is not used for cross-linkages. Another 35% of the -amino groups of lysine remain free. From the existing interpeptide chains 30% are not cross-linked.The addition of glycine to the minimal medium causes an increase of the glycine content in the murein, however, the extra alanine protion nearly disappears. The addition of serine leads to an increase of not only the serine portion but also the glycine portion in the murein. However, when alanine is added the alanine portion of murein is slightly increased and the glycine portion further decreased.The results of these experiments were compared to corresponding preliminary experiments with S. epidermidis (strain 66) and S. aureus (strain Copenhagen). In spite of modificative changes in the murein composition, clear genetical differences between the 3 strains were obvious.