Rapid superparamagnetic-beads-based automated immunoseparation of Zn-proteins from Staphylococcus aureus with nanogram yield.

Pathogenic bacteria have become a serious socio-economic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn-proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn-proteins by superparamagnetic beads modified with chicken anti-Zn(II) antibody. S. aureus culture was treated with ZnCl(2). Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle-surface interactions were tested and associated protein profiles were identified using SDS-PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS-PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.

Bio-sensing of cadmium(II) ions using Staphylococcus aureus.

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 µg mL(-1)) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds-specifically the protein metallothionein (0.79-26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190-5,827 µmol/min/mg of protein), and sulfhydryl groups (9.6-274.3 µmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, ß-glucosidase, phosphatase, N-acetyl ß-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components.

Tick-borne encephalitis and golden agers: position paper of the International Scientific Working Group on Tick-borne encephalitis (ISW-TBE).

One of the primary goals of the 11th Annual Meeting of the International Scientific Working Group on Tick-borne encephalitis (ISW-TBE) held in 2009 was to develop the first update of the Position Paper on TBE in Golden Agers, summarizing the most essential aspects of the disease in this age group. TBE morbidity has continued to increase in recent years, which is thought to be due to an interplay of social, political, ecological, economic and demographic factors combined with climate changes. Today's golden agers i.e. individuals aged 50 years or above, are healthier and more mobile, lead more active lifestyles and spend more time travelling and performing outdoor leisure activities. This places them at an increased risk of infection. At the same time, increasing age is associated with a quantitative and qualitative decline in innate and adaptive immunity, which is why elderly individuals are more susceptible to infection and severe disease than younger people. Also, their response to vaccination tends to be slower, antibody titres generally reach lower levels and titres tend to decrease earlier than in younger individuals. Evidence is accumulating that this is also the case with TBE vaccination, emphasizing the importance of administering the first TBE booster vaccination no later than 3 years after the completion of primary immunization or at an even shorter interval. Encouragingly, recent data have shown that the field effectiveness of TBE vaccination exceeds 97%, with no significant differences between age groups.