Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of beta-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost-and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.