Ethanol inactivation of orthonairoviruses in ixodid ticks.

Ixodid ticks represent vectors and reservoirs for a broad range of zoonotic pathogens. Collected ticks from field studies are therefore usually stored in ethanol, which in higher concentrations effectively inactivates most of the known tick-borne pathogens. Although commonly practiced as gold standard for inactivation, hardly any scientific data demonstrate that ethanol sufficiently penetrates the comparatively thick cuticula of ticks. Therefore, Amblyomma hebraeum tick pools were stored for 21 days in ethanol (96%). Afterwards, the ethanol was removed and the ticks were homogenized. Quantitative 1H-NMR spectroscopic analysis was applied to determine the residual concentration of ethanol inside the ticks. 1H-NMR spectroscopic analysis revealed that ethanol constituted 28.3-42.6 mg of the total weight of three ticks in the pools (89.9-121.5 mg). In addition, the low-pathogenic Hazara orthonairovirus (HAZV) was used as a cell culture model for this study. The virus was exposed to ethanol concentrations between 0 and 60% and incubated under various temperature conditions for four time periods. Afterwards, the residual virus infectivity was determined by titration. Following ethanol exposure, HAZV did not grow in cells after 9 h of exposure to an ethanol concentration of 25%. These results demonstrate an extremely low ethanol resistance of the virus, which was generally in line with previously reported ethanol inactivation data for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). After prolonged storage and impregnation, comparable ethanol concentrations are achieved in the ticks, indicating the suitability of this inactivation method also for Bunyaviruses in ticks. At the very least, a massive virus inactivation can be assumed. Definitive proof of virus inactivation would require a bioassay of ethanol-treated infected ticks under appropriate biosafety conditions.

Infection and metabolism - Streptococcus pneumoniae metabolism facing the host environment.

Streptococcus pneumoniae, a Gram-positive bacterium, can cause a broad range of severe diseases including pneumonia, septicemia and meningitis. The pneumococcal pathophysiology is highly dependent on host nutrients such as purines, pyrimidines, amino acids and carbon sources. The study of S. pneumoniae metabolism is essential for understanding the adaption strategies that are required to deal with the host environment during infection and to identify possible new drug targets. By using a combination of high analytical techniques, such as nuclear magnetic resonance (NMR), liquid chromatography (LC) and/or gas chromatography (GC) coupled with mass spectroscopy (MS) the metabolism of S. pneumoniae can be precisely investigated. This review summarizes the metabolism and major virulence factors of S. pneumoniae within the context of infection. Furthermore, it addresses the antibiotic resistance and its influence on metabolism as well as new antimicrobial substances for the treatment of S. pneumoniae.

In vivo tissue engineered bone versus autologous bone: stability and structure.

This pilot study investigated the biomechanical properties of prefabricated, vascularized bioartificial bone grafts, which may provide an alternative bone source for the restoration of segmental osseous defects. Vascularized bioartificial bone grafts comprise an artificial customized scaffold made of beta-tricalcium phosphate. Bone formation along the prefabricated scaffold is induced by autogenous cancellous bone. Vascularization of the bone graft is provided by the host's vascular system. Within 6 months, a mammalian bioreactor (sheep were used in the present study) creates heterotopic vascularized bioartificial bone grafts of a predetermined anatomical shape, which can be harvested for reconstructing osseous defects. The bioartificial bone grafts in this study contained up to 25% bone tissue, as shown by histomorphometric analysis and computed tomography. Moreover, unconfined compression tests revealed that the constructs had mechanical characteristics similar to those of ovine cancellous bone. Therefore, this method could be applied to generate vascularized prefabricated bone substitutes for critical-size defects.

GdpS contributes to Staphylococcus aureus biofilm formation by regulation of eDNA release.

In Staphylococcus aureus, the role of the GGDEF domain-containing protein GdpS remains poorly understood. Previous studies reported that gdpS mutant strains had decreased biofilm formation due to changes in icaADBC expression that were independent of cyclic-di-GMP levels. We deleted gdpS in three unrelated S. aureus isolates, and analyzed the resultant mutants for alterations in biofilm formation, metabolism and transcription. Dynamic imaging during biofilm development showed that GdpS inhibited early biofilm formation in only two out of the three strains examined, without affecting bacterial survival. However, quantification of biofilm formation using crystal violet staining revealed that inactivation of gdpS affected biofilm formation in all three studied strains. Extraction of metabolites from S. aureus cells confirmed the absence of cyclic-di-GMP, suggesting that biofilm formation in this species differs from that in other Gram-positive organisms. In addition, targeted mutagenesis demonstrated that the GGDEF domain was not required for GdpS activity. Transcriptomic analysis revealed that the vast majority of GGDEF-regulated genes were involved in virulence, metabolism, cell wall biogenesis and eDNA release. Finally, expression of lrgAB or deletion of cidABC in a strain lacking gdpS confirmed the role of GdpS on regulation of eDNA production that occurred without an increase in cell autolysis, but with a late increase in holin-mediated autolysis, in the presence of high oxacillin concentrations. In summary, S. aureus GdpS contributes to cell-to-cell interactions during early biofilm formation by influencing expression of lrgAB and cidABC mediated eDNA release. We conclude that GdpS acts as a negative regulator of eDNA release.