Single Bacterium Level Research Articles

Label-free identification of individual bacteria using Fourier transform light scattering.

Rapid identification of bacterial species is crucial in medicine and food hygiene. In order to achieve rapid and label-free identification of bacterial species at the single bacterium level, we propose and experimentally demonstrate an optical method based on Fourier transform light scattering (FTLS) measurements and statistical classification. For individual rod-shaped bacteria belonging to four bacterial species (Listeria monocytogenes, Escherichia coli, Lactobacillus casei, and Bacillus subtilis), two-dimensional angle-resolved light scattering maps are precisely measured using FTLS technique. The scattering maps are then systematically analyzed, employing statistical classification in order to extract the unique fingerprint patterns for each species, so that a new unidentified bacterium can be identified by a single light scattering measurement. The single-bacterial and label-free nature of our method suggests wide applicability for rapid point-of-care bacterial diagnosis.

Plasmonic imaging of protein interactions with single bacterial cells

Quantifying the interactions of bacteria with external ligands is fundamental to the understanding of pathogenesis, antibiotic resistance, immune evasion, and mechanism of antimicrobial action. Due to inherent cell-to-cell heterogeneity in a microbial population, each bacterium interacts differently with its environment. This large variability is washed out in bulk assays, and there is a need of techniques that can quantify interactions of bacteria with ligands at the single bacterium level. In this work, we present a label-free and real-time plasmonic imaging technique to measure the binding kinetics of ligand interactions with single bacteria, and perform statistical analysis of the heterogeneity. Using the technique, we have studied interactions of antibodies with single Escherichia coli O157:H7 cells and demonstrated a capability of determining the binding kinetic constants of single live bacteria with ligands, and quantify heterogeneity in a microbial population.

Structured attachment of bacterial molecular motors for defined microflow induction

AbstractBacterial rotational motor complexes that propel flagellated bacteria possess unique properties like their size of a few nanometres and the ability of selfreproduction that have led to various exciting applications including biohybrid nano-machines. One mandatory prerequisite to utilize bacterial nano motors in fluid applications is the ability to transfer force and torque to the fluid, which usually can be achieved by attachment of the bacterial cell to adequate surfaces. Additionally, for optimal transfer of force or torque, precise control of the position down to the single cell level is of utmost importance. Based on a PIV (particle image velocimetry) evaluation of the induced flow of single bacteria,we propose and demonstrate attachment of arbitrary patterns of motile bacterial cells in a fast light-based two-step process for the first time to our knowledge. First, these cells are pre-structured by holographic optical tweezers and then attached to a homogeneous, polystyrene-coated surface. In contrast to the few approaches that have been implemented up to now and which rely on pre-structured surfaces, our scheme allows for precise control on a single bacterium level, is versatile, interactive and has low requirements with respect to the surface preparation.

Single-Bacterial Profiling and Identification Based on Quantitative Phase Imaging

Rapid identification of bacterial species is crucial in medicine and food hygiene. For instance, bacterial invasion-related syndromes such as sepsis, pneumonia, abscess, meningitis, gastroenteritis and food poisoning require rapid cures which are appropriate for the invaded bacterial species. However, conventional culture-based or molecular-assay technologies do not match this time requirement in general, mainly due to their limited sensitivity. Here, we present the potential of quantitative phase imaging (QPI) techniques (1) for identification of bacterial species at single bacterium level. Employment of various QPI modalities provides the corresponding biophysical and biochemical properties of the bacterial cell in a rapid and label-free manner. First, we employed anisotropic Fourier transform light scattering (2) and demonstrated that light scattering from individual bacterium reflects structural information sufficient for identification of rod-shaped species. Second, the potential of spectroscopic QPI (3, 4) for extracting chemical composition of the bacterium with sub-genus sensitivity is suggested.1. Lee, K., K. Kim, J. Jung, J. H. Heo, S. Cho, S. Lee, G. Chang, Y. J. Jo, H. Park, and Y. K. Park. 2013. Quantitative phase imaging techniques for the study of cell pathophysiology: from principles to applications. Sensors 13:4170-4191.2. Kim, Y., J. M. Higgins, R. R. Dasari, S. Suresh, and Y. K. Park. 2012. Anisotropic light scattering of individual sickle red blood cells. J. Biomed. Opt. 17:040501.3. Park, Y., T. Yamauchi, W. Choi, R. Dasari, and M. S. Feld. 2009. Spectroscopic phase microscopy for quantifying hemoglobin concentrations in intact red blood cells. Optics Letters 34:3668-3670.4. Jang, Y., J. Jang, and Y. Park. 2012. Dynamic spectroscopic phase microscopy for quantifying hemoglobin concentration and dynamic membrane fluctuation in red blood cells. Optics Express 20.

Quorum Sensing and Self-Quorum Quenching in the Intracellular Pathogen Brucellamelitensis

Brucella quorum sensing has been described as an important regulatory system controlling crucial virulence determinants such as the VirB type IV secretion system and the flagellar genes. However, the basis of quorum sensing, namely the production of autoinducers in Brucella has been questioned. Here, we report data obtained from the use of a genetic tool allowing the in situ detection of long-chain N-acyl-homoserine lactones (AHL) activity at single bacterium level in Brucella melitensis. These data are consistent with an intrinsic production of AHL by B. melitensis in low concentration both during in vitro growth and macrophage infection. Moreover, we identified a protein, named AibP, which is homologous to the AHL-acylases of various bacterial species. In vitro and during infection, expression of aibP coincided with a decrease in endogenous AHL activity within B. melitensis, suggesting that AibP could efficiently impair AHL accumulation. Furthermore, we showed that deletion of aibP in B. melitensis resulted in enhanced virB genes expression and VirB8 production as well as in a reduced flagellar genes expression and production of FlgE (hook protein) and FliC (flagellin) in vitro. Altogether, these results suggest that AHL-dependent quorum sensing and AHL-quorum quenching coexist in Brucella, at least to regulate its virulence.

Probing single- to multi-cell level charge transport in Geobacter sulfurreducens DL-1

Microbial fuel cells, in which living microorganisms convert chemical energy into electricity, represent a potentially sustainable energy technology for the future. Here we report the single-bacterium level current measurements of Geobacter sulfurreducens DL-1 to elucidate the fundamental limits and factors determining maximum power output from a microbial fuel cell. Quantized stepwise current outputs of 92(±33) and 196(±20) fA are generated from microelectrode arrays confined in isolated wells. Simultaneous cell imaging/tracking and current recording reveals that the current steps are directly correlated with the contact of one or two cells with the electrodes. This work establishes the amount of current generated by an individual Geobacter cell in the absence of a biofilm and highlights the potential upper limit of microbial fuel cell performance for Geobacter in thin biofilms.

다공성 매질 표면에서 박테리아의 거동: 확산의 영향

It has been found that the colony size of bacteria grown on an agar plate decreases with increasing agar gel concentration. Evidenc from recent studies suggests that the bacterial colony dynamics is closely related with the mechanical properties of the substrate. We investigate whether bacterial growth on the agar substrate is controlled mostly by the nutrients' diffusion which is hindered more in porous medium than in solution. The number of bacterial cells in single colonies is found to be inversely correlated with agar concentration. High-resolution live cell imaging at the single bacterium level confirms that the bacterial growth rate is reduced with increasing agar concentration. There is a strong correlation between the slowed diffusion and the reduced number of cells in a high concentration of agar medium.

Photothermal antimicrobial nanotherapy and nanodiagnostics with self‐assembling carbon nanotube clusters

Unique properties of carbon nanotubes (CNTs) would open new avenues for addressing challenges to realize rapid and sensitive antimicrobial diagnostics and therapy for human pathogens. In this study, new CNTs' capabilities for photothermal (PT) antimicrobial nanotherapy were explored in vitro using Escherichia coli as a model bacterium. Single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) were incubated with E. coli K12 strain. CNTs' locations in bacteria and laser-induced thermal and accompanied effects around CNTs were estimated with TEM and PT microscopy, respectively. Multi-pulse lasers at 532 and 1064 nm with 12-ns pulse duration were used for irradiating sample mixtures at different laser fluences. Cell viability was evaluated using a bacterial viability test kit and epi-fluorescence microscopy. This study revealed CNTs' high binding affinity to bacteria, their capability to self-assemble as clusters at bacteria surfaces, and their inherent near-infrared (NIR) laser responsiveness. Cell viability was affected neither by CNTs alone nor by NIR irradiations alone. Notable changes in bacteria viability, caused by local thermal and accompanied bubble-formation phenomena, were observed starting at laser fluences of 0.1-0.5 J/cm(2) with complete bacteria disintegration at 2-3 J/cm(2) at both wavelengths. Furthermore, ethanol in reaction mixtures significantly (more than one order) enhanced bubble formation phenomena. This first application of laser-activated CNTs as PT contrast antimicrobial agents demonstrated its great potential to cause irreparable damages to disease-causing pathogens as well as to detect the pathogens at single bacterium level. This unique integration of laser and nanotechnology may also be used for drinking water treatment, food processing, disinfection of medical instrumentation, and purification of grafts and implants. Furthermore, the significant ethanol-induced enhancement of bubble formation provides another unique possibility to improve the efficiency of selective nanophotothermolysis for treating cancers, wounds, and vascular legions.