RESUMEN
Resistant bacteria are spreading worldwide, which makes fast antibiotic susceptibility testing and determination of the minimal inhibitory concentration (MIC) urgently necessary to select appropriate antibiotic therapy in time and, by this, improve patient's outcome and, at the same time, avoid inappropriate treatment as well as the unnecessary use of broad spectrum antibiotics that would foster further spread of resistant bacteria. Here, a simple and fast Raman spectroscopy-based procedure is introduced to identify antimicrobial susceptibilities and determine the MIC within only 2 h total analysis, marking a huge time savings compared to established phenotypic methods nowadays used in diagnostics. Sample preparation is fast and easy as well as comparable to currently established tests. The use of a dielectrophoresis chip allows automated collection of the bacteria in a micron-sized region for high-quality Raman measurement directly from bacterial suspensions. The new Raman spectroscopic MIC test was validated with 13 clinical E. coli isolates that show a broad range of ciprofloxacin resistance levels and were collected from patients with blood-stream infection. Micro-Raman spectroscopy was able to detect ciprofloxacin-induced changes in E. coli after only 90 min interaction time. Principal component analysis as well as a simple computed ratio of the Raman marker bands at 1458 and 1485 cm-1 show a clear concentration-dependent effect. The MIC values determined with the new Raman method are in good agreement with MICs obtained by reference methods (broth microdilution, Vitek-2, E-test) and can be used to provide a classification as sensitive, intermediate, or resistant using the clinical breakpoints provided by EUCAST.
Asunto(s)
Escherichia coli/clasificación , Pruebas de Sensibilidad Microbiana/métodos , Espectrometría Raman/métodos , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana/instrumentación , Análisis de Componente Principal , Espectrometría Raman/instrumentaciónRESUMEN
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.
Asunto(s)
Escherichia coli/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Pinzas Ópticas , Antibacterianos/efectos adversos , Escherichia coli/crecimiento & desarrollo , Micromanipulación/métodos , Análisis de Componente Principal , Espectrometría RamanRESUMEN
Vancomycin is an important glycopeptide antibiotic which is used to treat serious infections caused by Gram-positive bacteria. However, during the last years, a tremendous rise in vancomycin resistances, especially among Enterococci, was reported, making fast diagnostic methods inevitable. In this contribution, we apply Raman spectroscopy to systematically characterize vancomycin-enterococci interactions over a time span of 90 min using a sensitive Enterococcus faecalis strain and two different vancomycin concentrations above the minimal inhibitory concentration (MIC). Successful action of the drug on the pathogen could be observed already after 30 min of interaction time. Characteristic spectral changes are visualized with the help of multivariate statistical analysis (linear discriminant analysis and partial least squares regressions). Those changes were employed to train a statistical model to predict vancomycin treatment based on the Raman spectra. The robustness of the model was tested using data recorded by an independent operator. Classification accuracies of >90 % were obtained for vancomycin concentrations in the lower range of a typical trough serum concentration recommended for most patients during appropriate vancomycin therapy. Characterization of drug-pathogen interactions by means of label-free spectroscopic methods, such as Raman spectroscopy, can provide the knowledge base for innovative and fast susceptibility tests which could speed up microbiological analysis as well as finding applications in novel antibiotic screenings assays. Graphical Abstract E. faecalis is incubated with vancomycin and characterized by means of Raman spectroscopy after different time points. Characteristic spectral changes reveal efficient vancomycin-enterococci-interaction.
Asunto(s)
Antibacterianos/farmacología , Enterococcus faecalis/efectos de los fármacos , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana/métodos , Espectrometría Raman/métodos , Vancomicina/farmacología , HumanosRESUMEN
A Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements.
Asunto(s)
Cefalosporinas , Preparaciones Farmacéuticas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli , Fluoroquinolonas/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
In times of rising antibiotic resistances, there is a high need for fast, sensitive and specific methods to determine antibiotic susceptibilities of bacterial pathogens. Here, we present an integrated microfluidic device in which bacteria from diluted suspensions are captured in well-defined regions using on-chip dielectrophoresis and further analyzed in a label-free and non-destructive manner using Raman spectroscopy. Minimal sample preparation and automated sample processing ensure safe handling of infectious material with minimal hands-on time for the operator. Clinical applicability of the presented device is demonstrated by antibiotic susceptibility testing of Escherichia coli towards the commonly prescribed second generation fluoroquinolone ciprofloxacin. Ciprofloxacin resistant E. coli were differentiated from sensitive E. coli with high accuracy within roughly three hours total analysis time paving the way for future point-of-care devices. Spectral changes leading to the discrimination between sensitive and resistant bacteria are in excellent agreement with expected metabolic changes in the bacteria due to the mode of action of the drug. The robustness of the method was confirmed with experiments involving different chip devices with different designs, both electrode as well as microfluidics design, and material. Furthermore, general applicability was demonstrated with different operators over an extended time period of half a year.