Staphylococcus aureus uses IsdG and IsdI to convert heme into a mixture of staphylobilin isomers, 15-oxo-β-bilirubin and 5-oxo-δ-bilirubin, formaldehyde, and iron. The highly ruffled heme found in the heme-IsdI and IsdG complexes has been proposed to be responsible for the unique heme degradation products. We employed resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopies to examine the coordination and electronic structures of heme bound to IsdG and IsdI. Heme complexed to IsdG and IsdI is coordinated by a neutral histidine. The trans ligand is hydroxide in the ferric alkaline form of both proteins. In the ferric neutral form at pH 6.0, heme is six-coordinated with water as the sixth ligand for IsdG and is in the mixture of the five-coordinated and six-coordinated species for IsdI. In the ferrous CO-bound form, CO is strongly hydrogen bonded with a distal residue. The marker lines, ν2 and ν3, appear at frequencies that are distinct from other proteins having planar hemes. The EPR spectra for the ferric hydroxide and cyanide states might be explained by assuming the thermal mixing of the d-electron configurations, (dxy)2(dxz,dyz)3 and (dxz,dyz)4(dxy)1. The fraction for the latter becomes larger for the ferric cyanide form. In the ferric neutral state at pH 6.0, the quantum mechanical mixing of the high and intermediate spin configurations might explain the peculiar frequencies of ν2 and ν3 in the RR spectra. The heme ruffling imposed by IsdG and IsdI gives rise to unique electronic structures of heme, which are expected to modulate the first and subsequent steps of the heme oxygenation.
- MeSH
- bakteriální proteiny chemie MeSH
- elektronová paramagnetická rezonance MeSH
- hem chemie MeSH
- lidé MeSH
- oxid uhelnatý chemie MeSH
- oxygenasy se smíšenou funkcí chemie MeSH
- oxygenasy chemie MeSH
- Ramanova spektroskopie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus chemie MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- hem MeSH
- IsdG protein, Staphylococcus aureus MeSH Prohlížeč
- IsdI protein, Staphylococcus aureus MeSH Prohlížeč
- oxid uhelnatý MeSH
- oxygenasy se smíšenou funkcí MeSH
- oxygenasy MeSH
Bacteriophages, or "phages" for short, are viruses that replicate in bacteria. The therapeutic and biotechnological potential of phages and their lytic enzymes is of interest for their ability to selectively destroy pathogenic bacteria, including antibiotic-resistant strains. Introduction of phage preparations into medicine, biotechnology, and food industry requires a thorough characterization of phage-host interaction on a molecular level. We employed Raman tweezers to analyze the phage-host interaction of Staphylococcus aureus strain FS159 with a virulent phage JK2 (=812K1/420) of the Myoviridae family and a temperate phage 80α of the Siphoviridae family. We analyzed the timeline of phage-induced molecular changes in infected host cells. We reliably detected the presence of replicating phages in bacterial cells within 5 min after infection. Our results lay the foundations for building a Raman-based diagnostic instrument capable of real-time, in vivo, in situ, nondestructive characterization of the phage-host relationship on the level of individual cells, which has the potential of importantly contributing to the development of phage therapy and enzybiotics.
The formation of a hardly removable biofilm in food processing and clinical settings calls for a deeper understanding of composition of the matrix that protects the biofilm cells, as the crucial matrix component is extracellular DNA (eDNA), participating in adhesion, aggregation and penetration reduction, yet serving as a horizontal gene transfer reservoir. Therefore, we evaluated eDNA release from the biofilm of two pathogens, Listeria monocytogenes and Staphylococcus aureus, with respect to their origin under different culturing condition. Primarily, the biofilms were observed by confocal laser scanning microscopy (CLSM) under conditions mimicking the food processing environment and human body. The eDNA was quantitatively characterised based on its area by IMARIS. Next, the eDNA content and biofilm formation were quantified by spectrophotometry. Data from both sets of experiments were statistically evaluated. The eDNA release varied between the microorganism, culturing conditions and the origin of strains. Independent of the method used, the clinical strains of S. aureus released more eDNA than the food related strains at 37 °C. eDNA content can be crucial discriminating matrix component between food related and clinical strains. Deeper understanding of the eDNA role in such a phenomenon could facilitate the design of effective strategy for biofilm disruption.
- MeSH
- biofilmy * MeSH
- biologický transport MeSH
- DNA bakterií chemie genetika metabolismus MeSH
- extracelulární prostor mikrobiologie MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- Listeria monocytogenes chemie genetika fyziologie MeSH
- listeriové infekce mikrobiologie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus chemie genetika fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA bakterií MeSH
Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae are the most representative bacteria causing infectious diseases. Due to the increased application of antibiotics, the bacterial resistance is growing causing severe complications. Therefore, a sensitive determination of these pathogens is crucial for effective treatment. The aim of this study was to design an effective method for multiplex detection of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae taking advantage from properties of magnetic particles as well as fluorescent nanoparticles (quantum dots). The method was able to detect as low concentrations of bacteria as 102 CFU/mL using the bacteria-specific genes (fnbA, mecA and wcaG).
- Klíčová slova
- Barcode, Klebsiella pneumoniae, Magnetic particles, Methicillin-resistant Staphylococcus aureus, Quantum dots, Staphylococcus aureus,
- MeSH
- Klebsiella pneumoniae chemie genetika MeSH
- kvantové tečky chemie MeSH
- lidé MeSH
- mikrobiální testy citlivosti metody MeSH
- Staphylococcus aureus chemie genetika MeSH
- taxonomické DNA čárové kódování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Attachment of bacterial pathogens to the niche tissue in the host is the first step in biofilm formation leading to colonization and establishment of infection in the host. While the most common method used for determining the potential role of a bacterial antigen in biofilm formation has been demonstration of loss of this property using specific knockout mutants, it is an expensive and a laborious procedure. This study describes an alternative immunological assay for identification of attachment antigens of Staphylococcus aureus, potentially important in the development of an effective vaccine against infections caused by this pathogen. The method is based upon the concept of inhibition of attachment of S. aureus to PEGs coated with virulence antigen-specific antibodies. Antibodies used for validation of this assay were specific for ClfA, FnBPA, SdrD, PNAG and α-toxin, accredited biofilm-associated antigens of S. aureus.
- MeSH
- antigeny bakteriální analýza MeSH
- bakteriální adheze MeSH
- bakteriální adheziny analýza MeSH
- biofilmy růst a vývoj MeSH
- imunoanalýza metody MeSH
- polystyreny MeSH
- protilátky bakteriální metabolismus MeSH
- Staphylococcus aureus chemie fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny bakteriální MeSH
- bakteriální adheziny MeSH
- polystyreny MeSH
- protilátky bakteriální MeSH
The electro-osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro-osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused-silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV-visible detection. First the influence of the electro-osmotic flow on the peak shape of a marker of electro-osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical-water-treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 μg/mL albumin were reached with this method.
- Klíčová slova
- Capillary electrophoresis, Fused-silica capillaries, Supercritical water, Surface roughness gradient,
- MeSH
- albuminy chemie izolace a purifikace MeSH
- amfotericin B chemie izolace a purifikace MeSH
- elektroforéza kapilární přístrojové vybavení MeSH
- oxid křemičitý chemie MeSH
- Staphylococcus aureus chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- albuminy MeSH
- amfotericin B MeSH
- oxid křemičitý MeSH
The methicillin-resistant Staphylococcus aureus causes difficult-to-treat healthcare-associated infections in humans. For fast and effective selection of an appropriate antibiotic therapy, it is essential to have rapid and reliable methods for differentiation of methicillin-resistant S. aureus from less dangerous methicillin-sensitive S. aureus. There have been many methods for the identification of methicillin-resistant S. aureus described but none has been accepted as an international standard. The most commonly used techniques such as phenotyping and genotyping have a few disadvantages, for instance, these techniques are not reproducible and stable. In addition, they are time-consuming, expensive, and they are not capable to distinguish all S. aureus strains. In this study, the methicillin-resistant and methicillin-sensitive S. aureus isolates obtained from patients were extracted in hot water. The released proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing. These two methods were able to differentiate among tested bacterial strains. The proposed methods are time saving, they are applicable in standard biochemical laboratories, and they do not require any expensive equipment.
- MeSH
- bakteriologické techniky metody MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- isoelektrická fokusace metody MeSH
- lidé MeSH
- rezistence na methicilin * MeSH
- Staphylococcus aureus chemie klasifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The emergence of drug-resistant bacteria and new or changing infectious pathogens is an important public health problem as well as a serious socioeconomic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. Nanomaterial-based techniques including fluorescent labeling by quantum dots as well as immunoextraction by magnetic particles are excellent tools for such purposes. Moreover, the combination with capillary electrophoresis in miniaturized microchip arrangement brings numerous benefits such as fast and rapid analysis, low sample consumption, very sensitive electrochemical and fluorescent detection, portable miniaturized instrumentation, and rapid and inexpensive device fabrication. Here the use of superparamagnetic particle-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II)-containing proteins (Zn-proteins) is reported using a robotic pipetting system speeding up the sample preparation and enabling to analyze 48 real samples within 6 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with the sufficient yield for SDS-PAGE (several tens ng of proteins).
- MeSH
- bakteriální proteiny izolace a purifikace MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová metody MeSH
- imunomagnetická separace metody MeSH
- kvantové tečky * MeSH
- Staphylococcus aureus chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
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.
- MeSH
- bakteriální proteiny chemie izolace a purifikace metabolismus MeSH
- imunoglobulin G metabolismus MeSH
- imunomagnetická separace přístrojové vybavení metody MeSH
- kur domácí MeSH
- lidé MeSH
- limita detekce MeSH
- metaloproteiny chemie izolace a purifikace metabolismus MeSH
- protilátky bakteriální metabolismus MeSH
- robotika přístrojové vybavení metody MeSH
- Staphylococcus aureus chemie izolace a purifikace metabolismus MeSH
- zinek chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- imunoglobulin G MeSH
- metaloproteiny MeSH
- protilátky bakteriální MeSH
- zinek MeSH
Use of square-wave voltammetry (SWV) for determination of cefoperazone (CFPZ) in some buffers, bacterial culture, urine, and milk is described. CFPZ provides a specific voltammetric signal which is affected by pH and solution components. Determination of CFPZ in Britton-Robinson buffer, pH 4.4, is sensitive with a low detection limit (about 0.5 nmol L(-1)). In a more complex medium (bacterial 2YT medium, pH 7.2) the detection limit was approximately 1.5 micromol L(-1). We provide evidence that SWV is a suitable and quick method for CFPZ determination in a culture of living bacteria without separation of biomass. We have found big differences between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in cultivation in the presence of CFPZ, depending on time. When CFPZ is cleaved by penicillinase, a new SWV peak b appears at more positive potentials. This peak rises both with increasing concentration of enzyme and with cleavage time while the original CFPZ peak is simultaneously decreasing. We determined the concentration of CFPZ in the drug Pathozone by the standard addition method and achieved good agreement with the declared value of CFPZ in the drug. With a simple pretreatment procedure it is possible to determine CFPZ in milk; for urine no pretreatment was required. Using SWV we could detect CFPZ concentrations as low as 500 nmol L(-1) in bovine milk and human urine.
- MeSH
- antibakteriální látky analýza moč MeSH
- cefoperazon analýza moč MeSH
- elektrochemie metody MeSH
- kultivační média MeSH
- léčivé přípravky chemie MeSH
- mikrobiální testy citlivosti MeSH
- mléko chemie MeSH
- Staphylococcus aureus chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- cefoperazon MeSH
- kultivační média MeSH
- léčivé přípravky MeSH
