RESUMO
The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony's unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern. KEY POINTS: ⢠Individual bacteria cells are placed on gradient agar plates by a cell sorter ⢠Laser-light scatter patterns are used to recognize antibiotic-resistant organisms ⢠Scatter patterns formed by colonies correspond to AMR-associated phenotypes.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Fenótipo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/classificação , Citometria de Fluxo/métodos , Testes de Sensibilidade Microbiana/métodos , LuzRESUMO
Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment.
Assuntos
Bioensaio/métodos , Citometria de Fluxo/métodos , Bioensaio/tendências , Análise de Dados , Citometria de Fluxo/tendências , Humanos , Software/tendênciasRESUMO
Despite recent progress in cell-analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate-up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8-15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well-separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next-generation flow cytometer based on a 32-channel PMT array detector, a phase-volume holographic grating, and a fast electronic board. The system is capable of full-spectral data collection and spectral analysis at the single-cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in <5 µs. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data-collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification.
Assuntos
Citometria de Fluxo/instrumentação , Análise de Célula Única/instrumentação , Animais , Anticorpos Monoclonais/química , Citometria de Fluxo/métodos , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Análise de Célula Única/métodosRESUMO
Biological microparticles, including bacteria, scatter light in all directions when illuminated. The complex scatter pattern is dependent on particle size, shape, refraction index, density, and morphology. Commercial flow cytometers allow measurement of scattered light intensity at forward and perpendicular (side) angles (2 degrees Assuntos
Bactérias/citologia
, Citometria de Fluxo/métodos
, Inteligência Artificial
, Automação
, Bacillus subtilis/metabolismo
, Bactérias/metabolismo
, Enterococcus faecalis/metabolismo
, Desenho de Equipamento
, Escherichia coli/metabolismo
, Listeria/metabolismo
, Modelos Estatísticos
, Reconhecimento Automatizado de Padrão
, Reprodutibilidade dos Testes
, Espalhamento de Radiação
RESUMO
The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.
Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oniocompostos/farmacologia , Superóxidos/metabolismo , Animais , Antioxidantes/farmacologia , Caspase 3 , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Ativação Enzimática/efeitos dos fármacos , Fibrossarcoma , Expressão Gênica , Células HL-60 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Mitocôndrias Cardíacas/ultraestrutura , Miocárdio/ultraestrutura , Ratos , Partículas Submitocôndricas/metabolismo , Superóxido Dismutase/genética , Superóxidos/farmacologia , Células Tumorais CultivadasRESUMO
The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX-Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX-Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX-Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference.
Assuntos
Citometria de Fluxo/métodos , Mitocôndrias/metabolismo , Neutrófilos/metabolismo , Fagossomos/metabolismo , Ácidos/metabolismo , Adulto , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Feminino , Corantes Fluorescentes , Humanos , Lisossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Fagocitose/imunologia , Fagossomos/imunologia , Explosão Respiratória/imunologia , Coloração e Rotulagem , Zimosan/metabolismoRESUMO
We have developed an automated system for drug screening using a single-cell-multiple functional response technology. The approach uses a semiautomated preparatory system, high-speed sample collection, and a unique analytical tool that provides instantaneous results for compound dilutions using 384-well plates. The combination of automation and rapid robotic sampling increases quality control and robustness. High-speed flow cytometry is used to collect single-cell results together with a newly defined analytical tool for extraction of IC(50) curves for multiple assays per cell. The principal advantage is the extreme speed of sample collection, with results from a 384-well plate being completed for both collection and data processing in less than 10 min. Using this approach, it is possible to extract detailed drug response information in a highly controlled fashion. The data are based on single-cell results, not populations. With simultaneous assays for different functions, it is possible to gain a more detailed understanding of each drug/compound interaction. Combined with integrated advanced data processing directly from raw data files, the process from sampling to analytical results is highly intuitive. Direct PubMed links allow review of drug structure and comparisons with similar compounds.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Análise de Célula Única/métodos , Automação , Citometria de Fluxo , Células HL-60 , Humanos , Concentração Inibidora 50 , Mitocôndrias/metabolismo , Fatores de TempoRESUMO
We describe a model-based instrument design combined with a statistical classification approach for the development and realization of high speed cell classification systems based on light scatter. In our work, angular light scatter from cells of four bacterial species of interest, Bacillus subtilis, Escherichia coli, Listeria innocua, and Enterococcus faecalis, was modeled using the discrete dipole approximation. We then optimized a scattering detector array design subject to some hardware constraints, configured the instrument, and gathered experimental data from the relevant bacterial cells. Using these models and experiments, it is shown that optimization using a nominal bacteria model (i.e., using a representative size and refractive index) is insufficient for classification of most bacteria in realistic applications. Hence the computational predictions were constituted in the form of scattering-data-vector distributions that accounted for expected variability in the physical properties between individual bacteria within the four species. After the detectors were optimized using the numerical results, they were used to measure scatter from both the known control samples and unknown bacterial cells. A multivariate statistical method based on a support vector machine (SVM) was used to classify the bacteria species based on light scatter signatures. In our final instrument, we realized correct classification of B. subtilis in the presence of E. coli,L. innocua, and E. faecalis using SVM at 99.1%, 99.6%, and 98.5%, respectively, in the optimal detector array configuration. For comparison, the corresponding values for another set of angles were only 69.9%, 71.7%, and 70.2% using SVM, and more importantly, this improved performance is consistent with classification predictions.
Assuntos
Bacillus subtilis/classificação , Técnicas de Tipagem Bacteriana/métodos , Enterococcus faecalis/classificação , Escherichia coli/classificação , Listeria/classificação , Modelos Biológicos , Espalhamento de Radiação , Inteligência Artificial , Bacillus subtilis/citologia , Bacillus subtilis/isolamento & purificação , Biometria , Enterococcus faecalis/citologia , Enterococcus faecalis/isolamento & purificação , Desenho de Equipamento , Escherichia coli/citologia , Escherichia coli/isolamento & purificação , Citometria de Fluxo , Luz , Listeria/citologia , Listeria/isolamento & purificação , Análise Multivariada , Reconhecimento Automatizado de Padrão/métodos , RefratometriaRESUMO
Nanoparticles and bacteria can be used, independently, to deliver genes and proteins into mammalian cells for monitoring or altering gene expression and protein production. Here, we show the simultaneous use of nanoparticles and bacteria to deliver DNA-based model drug molecules in vivo and in vitro. In our approach, cargo (in this case, a fluorescent or a bioluminescent gene) is loaded onto the nanoparticles, which are carried on the bacteria surface. When incubated with cells, the cargo-carrying bacteria ('microbots') were internalized by the cells, and the genes released from the nanoparticles were expressed in the cells. Mice injected with microbots also successfully expressed the genes as seen by the luminescence in different organs. This new approach may be used to deliver different types of cargo into live animals and a variety of cells in culture without the need for complicated genetic manipulations.
Assuntos
DNA/administração & dosagem , DNA/genética , Portadores de Fármacos/química , Nanopartículas/química , Transfecção/métodos , Transformação Bacteriana/genética , Teste de MateriaisRESUMO
Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO(2) supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.
Assuntos
Bacillus cereus/metabolismo , Técnicas Biossensoriais/métodos , Hibridomas/citologia , Listeria monocytogenes/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Dióxido de Carbono/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Hibridomas/efeitos dos fármacos , Hibridomas/microbiologia , Concentração de Íons de Hidrogênio , Listeria monocytogenes/crescimento & desenvolvimento , Camundongos , Fatores de TempoRESUMO
Scatter of a two-dimensional Gaussian beam of a rectangular cross section by individual particles suspended in a fluid in a cylindrical channel is modeled by using a full-wave approach. First, the internal and scattered fields associated with the cylindrical channel and the two-dimensional Gaussian beam are computed. The spatial variations of the computed electromagnetic field inside the channel indicate that particles and cells of sizes relevant to flow cytometry are subjected to essentially plane-wave illumination, and hence Lorenz-Mie theory is applicable for spherical particles. Further, it is assumed that the perturbation of the electromagnetic field in the channel that is due to the presence of a particle is negligible, allowing us to ignore the interactive scatter of the particle and the channel (they are electromagnetically uncoupled). This approximation is valid when the particle intercepts a small fraction of the total energy inside the channel and when the particle or cell has a low relative refractive index. Measurements of scatter from the channel agree with the analytical model and are used to determine the location of detectors to measure scatter from particles in the channel. Experimental results of accumulated scatter from single latex spheres flowing in the channel show good agreement with computed results, thereby validating the internal field and uncoupled scatter models.
RESUMO
Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.