The search for the "Holy Grail" in clinical diagnostic microbiology-a reliable, accurate, low-cost, real-time, easy-to-use method-has brought up several methods with the potential to meet these criteria. One is Raman spectroscopy, an optical, nondestructive method based on the inelastic scattering of monochromatic light. The current study focuses on the possible use of Raman spectroscopy for identifying microbes causing severe, often life-threatening bloodstream infections. We included 305 microbial strains of 28 species acting as causative agents of bloodstream infections. Raman spectroscopy identified the strains from grown colonies, with 2.8% and 7% incorrectly identified strains using the support vector machine algorithm based on centered and uncentred principal-component analyses, respectively. We combined Raman spectroscopy with optical tweezers to speed up the process and captured and analyzed microbes directly from spiked human serum. The pilot study suggests that it is possible to capture individual microbial cells from human serum and characterize them by Raman spectroscopy with notable differences among different species. IMPORTANCE Bloodstream infections are among the most common causes of hospitalizations and are often life-threatening. To establish an effective therapy for a patient, the timely identification of the causative agent and characterization of its antimicrobial susceptibility and resistance profiles are essential. Therefore, our multidisciplinary team of microbiologists and physicists presents a method that reliably, rapidly, and inexpensively identifies pathogens causing bloodstream infections-Raman spectroscopy. We believe that it might become a valuable diagnostic tool in the future. Combined with optical trapping, it offers a new approach where the microorganisms are individually trapped in a noncontact way by optical tweezers and investigated by Raman spectroscopy directly in a liquid sample. Together with the automatic processing of measured Raman spectra and comparison with a database of microorganisms, it makes the whole identification process almost real time.
- MeSH
- Algorithms MeSH
- Humans MeSH
- Optical Tweezers MeSH
- Pilot Projects MeSH
- Spectrum Analysis, Raman * methods MeSH
- Sepsis * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Form and function of the mitotic spindle depend on motor proteins that crosslink microtubules and move them relative to each other. Among these are kinesin-14s, such as Ncd, which interact with one microtubule via their non-processive motor domains and with another via their diffusive tail domains, the latter allowing the protein to slip along the microtubule surface. Little is known about the influence of the tail domains on the protein's performance. Here, we show that diffusive anchorage of Ncd's tail domains impacts velocity and force considerably. Tail domain slippage reduced velocities from 270 nm s-1 to 60 nm s-1 and forces from several piconewtons to the sub-piconewton range. These findings challenge the notion that kinesin-14 may act as an antagonizer of other crosslinking motors, such as kinesin-5, during mitosis. It rather suggests a role of kinesin-14 as a flexible element, pliantly sliding and crosslinking microtubules to facilitate remodeling of the mitotic spindle.
- MeSH
- Spindle Apparatus metabolism MeSH
- Kinesins isolation & purification metabolism MeSH
- Microtubules metabolism MeSH
- Mitosis physiology MeSH
- Optical Tweezers MeSH
- Protein Domains MeSH
- Microtubule-Associated Proteins genetics isolation & purification metabolism MeSH
- Drosophila Proteins isolation & purification metabolism MeSH
- Recombinant Proteins genetics isolation & purification metabolism MeSH
- Saccharomyces cerevisiae Proteins genetics isolation & purification metabolism MeSH
- Protein Binding physiology MeSH
- Green Fluorescent Proteins genetics isolation & purification metabolism MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.
A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.
- MeSH
- Algorithms MeSH
- Principal Component Analysis MeSH
- Bacteria metabolism MeSH
- Biofilms * MeSH
- Cell Adhesion MeSH
- Phagocytosis MeSH
- Bacterial Physiological Phenomena MeSH
- Optical Tweezers * MeSH
- Polysaccharides chemistry MeSH
- Spectrum Analysis, Raman * MeSH
- Staphylococcus epidermidis metabolism MeSH
- Hot Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in that they have strong light absorption in the visible range, and by the fact that their reaction centers are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux density caused photodamage that was strongly wavelength dependent. The photochemical activity before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30s exposure to incident radiation at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable photodamage.
Optical trapping of particles has become a powerful non-mechanical and non-destructive technique for precise particle positioning. The manipulation of particles in the evanescent field of a channel waveguide potentially allows for sorting and trapping of several particles and cells simultaneously. Channel waveguide designs can be further optimized to increase evanescent field prior to the fabrication process. This is crucial in order to make sure that the surface intensity is sufficient for optical trapping. Simulation configurations are explained in detail with specific simulation flow. Discussion on parameters optimization; physical geometry, optical polarization and wavelength is included in this paper. The effect of physical, optical parameters and beam spot size on evanescent field has been thoroughly discussed. These studies will continue toward the development of a novel copper ion-exchanged waveguide as a method of particle sorting, with biological cell propulsion studies presently underway.
- MeSH
- Equipment Failure Analysis MeSH
- Computer-Aided Design MeSH
- Equipment Design MeSH
- Ions chemistry MeSH
- Copper chemistry MeSH
- Optical Tweezers * MeSH
- Computer Simulation MeSH
- Surface Plasmon Resonance instrumentation MeSH
- Scattering, Radiation MeSH
- Light MeSH
- Models, Theoretical * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.
- MeSH
- Adenosine Triphosphatases metabolism MeSH
- Amino Acid Motifs MeSH
- Biological Transport MeSH
- Biological Assay MeSH
- DNA Helicases chemistry MeSH
- DNA chemistry MeSH
- Endonucleases chemistry MeSH
- Escherichia coli enzymology MeSH
- Kinetics MeSH
- Molecular Motor Proteins chemistry metabolism MeSH
- Molecular Sequence Data MeSH
- Mutagenesis MeSH
- Mutant Proteins chemistry metabolism MeSH
- Optical Tweezers MeSH
- Protein Subunits chemistry metabolism MeSH
- Deoxyribonucleases, Type I Site-Specific chemistry metabolism MeSH
- Amino Acid Sequence MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
