14 research outputs found
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Predictive Modeling of Antibiotic Susceptibility in E. Coli Strains Using the U-Net Network and One-Class Classification
The antibiotic resistance of bacterial pathogens has become one of the most serious global health issues due to misusing and overusing of antibiotics. Recently, different technologies were developed to determine bacteria susceptibility towards antibiotics; however, each of these technologies has its advantages and limitations in clinical applications. In this contribution, we aim to assess and automate the detection of bacterial susceptibilities towards three antibiotics; i.e. ciprofloxacin, cefotaxime and piperacillin using a combination of image processing and machine learning algorithms. Therein, microscopic images were collected from different E. coli strains, then the convolutional neural network U-Net was implemented to segment the areas showing bacteria. Subsequently, the encoder part of the trained U-Net was utilized as a feature extractor, and the U-Net bottleneck features were utilized to predict the antibiotic susceptibility of E. coli strains using a one-class support vector machine (OCSVM). This one-class model was always trained on images of untreated controls of each bacterial strain while the image labels of treated bacteria were predicted as control or non-control images. If an image of treated bacteria is predicted as control, we assume that these bacteria resist this antibiotic. In contrast, the sensitive bacteria show different morphology of the control bacteria; therefore, images collected from these treated bacteria are expected to be classified as non-control. Our results showed 83% area under the receiver operating characteristic (ROC) curve when OCSVM models were built using the U-Net bottleneck features of control bacteria images only. Additionally, the mean sensitivities of these one-class models are 91.67% and 86.61% for cefotaxime and piperacillin; respectively. The mean sensitivity for the prediction of ciprofloxacin is only 59.72% as the bacteria morphology was not fully detected by the proposed method
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Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress
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
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Raman spectroscopy follows time-dependent changes in T lymphocytes isolated from spleen of endotoxemic mice
T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hostsâ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hostsâ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult
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Towards Bacteria Counting in DI Water of Several Microliters or Growing Suspension Using Impedance Biochips
We counted bacterial cells of E. coli strain K12 in several-microliter DI water or in several-microliter PBS in the low optical density (OD) range (OD = 0.05â1.08) in contact with the surface of Si-based impedance biochips with ring electrodes by impedance measurements. The multiparameter fit of the impedance data allowed calibration of the impedance data with the concentration cb of the E. coli cells in the range of cb = 0.06 to 1.26 Ă 109 cells/mL. The results showed that for E. coli in DI water and in PBS, the modelled impedance parameters depend linearly on the concentration of cells in the range of cb = 0.06 to 1.26 Ă 109 cells/mL, whereas the OD, which was independently measured with a spectrophotometer, was only linearly dependent on the concentration of the E. coli cells in the range of cb = 0.06 to 0.50 Ă 109 cells/mL
Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells
In recent years, human dendritic cells (DCs) could be subdivided into CD304+ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c+, CD16+, and CD141+ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141+ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca2+ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141+ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8+ T cells when directly compared with CD1c+ DCs, CD16+ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141+ DCs correspond to mouse CD8+ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141+ DCs as professional antigen cross-presenting DCs in the human
Mechanisms generating biliary lipid specificity
Die vorliegende Arbeit beschĂ€ftigt sich mit den molekularen Prozessen der Lipidanreicherung in der GallenflĂŒssigkeit. Leberzellen (Hepatozyten) sind polare Zellen, die fĂŒr die Sekretion der GallenflĂŒssigkeit verantwortlich sind. Die Anbindung an den Blutkreislauf besteht ĂŒber die basolaterale Membran. Durch die gegenĂŒberliegende, sogenannte apikale Membran werden zwischen benachbarten Leberzellen tubulĂ€re Stukturen (bile canaliculi, BC) gebildet, in die die GallenflĂŒssigkeit abgesondert wird. Daher wird diese Membran auch als Canalicularmembran (CM) bezeichnet. Die GallenflĂŒssigkeit besitzt hinsichtlich ihrer Lipidzusammensetzung eine bemerkenswerte SpezifitĂ€t. Obwohl der Anteil von Phosphatidylcholin (PC) an den Phospholipiden der CM nur 35% betrĂ€gt, macht es 95% der Phospholipide der GallenflĂŒssigkeit aus. Mögliche Mechanismen, die zur SpezifitĂ€t der Lipidsekretion in die GallenflĂŒssigkeit fĂŒhren, werden untersucht und diskutiert. Phospholipide werden aus der Ă€uĂeren Lamelle der CM durch Gallensalze herausgelöst. Die Wechselwirkung von Gallensalzen mit Phospholipiden ist kopfgruppenunspezifisch. Eine Solubilisierung von Phosphatidylserin (PS) und Phosphatidylethanolamin (PE) durch Gallensalze könnte durch die Wirkung einer Aminophospholipidtranslokase (APLT) verhindert werden, die diese Lipide aktiv auf die zytoplasmatische Seite der Membran pumpt. Zur ĂberprĂŒfung dieser Hypothese wurden Versuche durchgefĂŒhrt, um die AktivitĂ€t einer APLT in der CM nachzuweisen. Dabei wurde die Hepatomazelllinie HepG2 eingesetzt, die in der Lage ist, Canalicularvakuolen (BC) zu bilden. ZunĂ€chst wurde die EinwĂ€rtsbewegung einer Reihe fluoreszierender Lipidanaloga mit unterschiedlicher AffinitĂ€t zur APLT charakterisiert. Dies geschah an der basolateralen Membran von HepG2 Zellen, wo eine APLT-AktivitĂ€t bereits bekannt ist. Die Aufnahme geeigneter APLT-Substrate konnte durch den APLT-Inhibitor Suramin reduziert werden. Ebenso wurde die AffinitĂ€t eines Paares von PS-Analoga bestĂ€tigt, von denen Diether PS ein "schlechtes" und Diacyl PS ein "gutes" APLT-Substrat darstellt. Im zweiten Schritt wurde die Anreicherung der gleichen Analoga in BC von HepG2 Zellen untersucht. Es ergab sich eine auffallende Korrelation zwischen einer APLT vermittelten Aufnahme von Phospholipidanaloga an der basolateralen Membran und dem Fehlen dieser Analoga im Lumen der BC. Wenn Zellen mit Phospholipiden markiert wurden, die keine oder nur "schlechte" APLT-Substrate darstellen, erschienen die BC stark fluoreszierend. Diese Beobachtungen zeigen, dass eine APLT-AktivitĂ€t in der CM von Hepatozyten vorhanden ist, welche das Fehlen der Aminophospholipide in der GallenflĂŒssigkeit erklĂ€rt. Ein zweiter Schwerpunkt dieser Arbeit war die Untersuchung der Rolle von MDR-Proteinen (wie MDR3) bei der Lipidsekretion in die GallenflĂŒssigkeit. Aufgrund bisheriger Arbeiten wird vermutet, dass MDR3 daran als spezifischer Membrantransporter fĂŒr PC beteiligt ist. In der vorliegenden Arbeit konnte jedoch gezeigt werden, dass verschiedene MDR-Inhibitoren die Anreicherung fluoreszierender Phospholipidanaloga in den BC von HepG2 Zellen nur wenig reduzieren. Diese Beobachtung kann unter der Annahme erklĂ€rt werden, dass MDR3 eher fĂŒr die Exposition von PC an der lumenalen Seite der CM verantwortlich ist, als fĂŒr den Tranport von PC ĂŒber die Membran. Solche "Liftase"-AktivitĂ€t von MDR3 könnte endogenes PC der Detergenzwirkung von Gallensalzen zugĂ€nglich machen, ein Prozess, der fĂŒr die hydrophileren fluoreszierenden PC-Analoga nicht nötig ist. Im dritten Teil wird die Rolle von Sphingolipiden und die Bildung von "Rafts" in der CM behandelt. Solche MembrandomĂ€nen sollten die Solubilisierung von Spingolipiden in die GallenflĂŒssigkeit verhindern. Eine Anreicherung fluoreszierender Sphingolipidanaloga in den BC wurde jedoch nachgewiesen, was darauf hindeutet, dass die verwendeten Analoga das Verhalten endogener Sphingolipide in der CM nicht korrekt wiederspiegeln. Im abschlieĂenden Teil dieser Arbeit wurden die Grundlagen fĂŒr eine Methode zur AufklĂ€rung der physikochemischen Prozesse der Lipidsekretion an der Canalicularmembran gelegt. Die starke UmgebungsabhĂ€ngigkeit der Fluoreszenzlebensdauer fĂŒr verschiedene fluoreszierende Lipidanaloga wurde in einer Reihe von Modellumgebungen analysiert und deren Nutzbarkeit fĂŒr die Vorhersage der Lipidorganisation geprĂŒft. Insbesondere wurde die Wechselwirkung verschiedener Gallensalze mit Lipidanaloga und der Fluoreszenzresonanzenergietransfer zwischen verschiedenen Lipidanaloga charakterisiert. Diese Daten sind Ausgangsbasis fĂŒr die mikroskopische Charakterisierung der Organisation von Lipidanaloga in den BC in vivo.This thesis addresses the molecular processes which are important in the formation of bile fluid. The polar liver cells (hepatocytes) secrete the bile fluid at their apical (canalicular) membrane into tubular bile canaliculi (BC) which are formed between adjacent cells. The basolateral membrane of hepatocytes faces the blood vessel. Bile fluid possesses a remarkable specificity regarding its lipid composition. Even though phosphatidylcholine (PC) contributes to only 35% of the phospholipids in the canalicular membrane, it constitutes 95% of biliary phospholipids. In this thesis possible mechanism that might lead to the specificity in biliary lipid secretion are analysed and discussed. Phospholipids are secreted from the outer leaflet of the canalicular membrane into bile by the effect of bile salts. The interaction of bile salts with phospholipids was shown to be independent of the phospholipid headgroup. Solubilisation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by bile salts could be prevented by the action of an aminophospholipid translocase (APLT) which actively pumps these lipids to the cytoplasmic leaflet of the membrane. Experiments to demonstrate a canalicular APLT activity were performed to proof this hypothesis. For this, the hepatoma cell line HepG2 which is able to polarise and to form a canalicular vacuole (BC) was utilised. A panel of fluorescent lipid analogues with different affinities to this transporter was used and first characterised at the basolateral membrane of HepG2 cells, where an APLT activity was already demonstrated. The rapid APLT mediated uptake of aminophospholipid analogues representing appropriate substrates of APLT was reduced by applying the inhibitor suramin. The affinity of a pair of PS analogues with diether NBD-PS as a poor APLT substrate and diacyl NBD-PS representing a suitable substrate was confirmed. In a next step the enrichment of the same phospholipid analogues in the BC was investigated. There was a striking correlation between APLT mediated uptake of phospholipid analogues at the basolateral membrane and absence of these analogues from the BC. In the case of phospholipid analogues that were no or poor substrates of APLT the BC appeared highly fluorescent, indicating that indeed a canalicular APLT is responsible and sufficient for biliary absence of aminophospholipids. Further experiments were aimed on the investigation of the role of MDR proteins (as MDR3) in biliary lipid secretion. It has been proposed that MDR3, which is crucial for biliary phospholipid secretion, acts as a specific flippase for PC. However, different MDR inhibitors did not completely abolish the enrichment of fluorescent phospholipid analogues in the BC in this study. This observation can be explained assuming that MDR3 is responsible for the exposure of PC at the lumenal side of the canalicular membrane rather than for its transport across the membrane. Such a "liftase" activity of MDR could make endogenous PC accessible to the detergent bile salts which is not necessary for its more hydrophilic fluorescent analogues. The third part of this thesis addressed the role of sphingolipids and the formation of detergent resistant rafts in the canalicular membrane. Rafts are thought to prevent sphingolipid solubilisation into bile. Fluorescent sphingolipid analogues were found to enrich in the BC even at low temperatures, however. These experiments suggest that the applied analogues might not suitably represent the majority of sphingolipids in the canalicular membrane. The final part of this study provides the basis for a method to investigate the physico-chemical processes occurring during lipid secretion at the canalicular membrane. The sensitivity of fluorescence life times on environmental changes was analysed using fluorescent lipid analogues in a set of model environments and its utility for predicting biliary lipid organisation is discussed. Especially the interaction of different bile salts with lipid analogues and fluorescence energy transfer between distinct lipid analogues was characterised. These data can be utilised for characterisation of the organisation of biliary enriched lipid analogues in vivo at a microscopic level in future
Microfluidic System for Cell Mixing and Particle Focusing Using Dean Flow Fractionation
Recent developments in the field of additive manufacturing processes have led to tremendous technological progress and opened directions for the field of microfluidics. For instance, new flexible materials for 3D printing allow the substitution of polydimethylsiloxane (PDMS) in microfluidic prototype development. Three-dimensional-printed microfluidic components open new horizons, in particular for the automated handling of biological cells (e.g., eukaryotic cells or bacteria). Here, we demonstrate how passive mixing and passive separation processes of biological cells can be realized using 3D printing concepts for rapid prototyping. This technique facilitates low-cost experimental setups that are easy to modify and adopt for specific detection and diagnostic purposes. In particular, printing technologies based on fused deposition modeling and stereolithography are used and their realization is discussed. Additive technologies enable the fabrication of multiplication mixers, which overcome shortcomings of current pillar or curve-based techniques and enable efficient mixing, also of biological cells without affecting viability. Using standard microfluidic components and state-of-the art 3D printing technologies, we realize a separation system based on Dean flow fragmentation without the use of PDMS. In particular, we describe the use of a 3D-printed helix for winding a capillary for particle flow and a new chip design for particle separation at the outlet. We demonstrate the functionality of the system by successful isolation of ~12 ”m-sized particles from a particle mixture containing large (~12 ”m, typical size of eukaryotic cells) and small (~2 ”m, typical size of bacteria or small yeasts) particles. Using this setup to separate eukaryotic cells from bacteria, we could prove that cell viability is not affected by passage through the microfluidic systems
Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress
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
Performance comparison of Findaures and Otsuâs thresholding method on simulated infected mouse bone tissue images (Accuracy, Precision, Sensitivity, and F1 Score) across four trials.
Performance comparison of Findaures and Otsuâs thresholding method on simulated infected mouse bone tissue images (Accuracy, Precision, Sensitivity, and F1 Score) across four trials.</p