Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy.

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix. Graphical abstract.

Bacterial phenotype dependency from CO2 measured by Raman spectroscopy.

In recent years, Raman spectroscopy has become an established method to study medical, biological or environmental samples. Since Raman spectroscopy is a phenotypic method, many parameters can influence the spectra. One of these parameters is the concentration of CO2, as this never remains stable in nature, but always adjusts itself in a dynamic equilibrium. So, it is obvious that the concentration of CO2 cannot be controlled but it might have a big impact on the bacteria and bacterial composition in medical samples. When using a phenotypic method like Raman spectroscopy it is also important to know the influence of CO2 to the dataset. To investigate the influence of CO2 towards Raman spectra we cultivated E. coli at different concentration of CO2 since this bacterium is able to switch metabolism from aerobic to microaerophilic conditions. After applying statistic methods small changes in the spectra became visible and it was even possible to observe the change of metabolism in this species according to the concentration of CO2.

Simulation of Transportation and Storage and Their Influence on Raman Spectra of Bacteria.

Recently, Raman spectroscopy has become more and more in the focus of bacterial identification as it is a culture-independent, nondestructive, and contact-less method. Since Raman spectroscopy is a phenotypic method, lots of parameters can influence the spectra. One of the least controllable factors is transport and storage, but it is often not taken into account, and therefore these influences on the Raman spectra of bacteria are unknown. In order to investigate this effect, we simulated the transport and storage of bacteria under different conditions and investigated them with Raman spectroscopy. With a look at the mean spectra, only one bacterium showed differences during the storage conditions. However, after applying chemometric methods, changes in the data could be found within all bacteria during storage times. This study shows how drastic the effect will influence a database, depending on the different handling or storage. Therefore, it is of the utmost importance to consider these nonbiological influences when planning further experiments and evaluating the resulting data.

Simultaneous Quantification of Nine Antimicrobials by LC-MS/MS for Therapeutic Drug Monitoring in Critically Ill Patients.

BACKGROUND: Adequate antibiotic treatment is a prerequisite for the successful treatment of systemic infections. Based on accumulating scientific evidence, a fixed dosage regimen can lead to insufficient and ineffective antibiotic therapy. Thus, the aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of antimicrobials by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for the development of personalized therapy regimens using therapeutic drug monitoring. METHODS: A method was developed for the simultaneous quantification of 9 antimicrobials (aciclovir, ampicillin, cefuroxime, ciprofloxacin, meropenem, metronidazole, piperacillin, rifampicin, and tazobactam) in lithium-heparin plasma. A simple sample preparation method and a chromatographic run time of 10 minutes enabled the quick processing of the samples. The method was validated according to the guidelines for bioanalytical method validation of the European Medicines Agency and addressed sensitivity, specificity, linearity, accuracy, precision, dilution integrity, carry-over, recovery, matrix effects, and stability. RESULTS: The chromatographic run time was 10 minutes and antimicrobials eluted at retention times ranging from 1.1 to 2.2 minutes. Calibration curve for all antimicrobials was linear over a range of 1-100 mg/L, and a 2-fold or 5-fold dilution of the samples was possible. The method accuracy ranged from 85.1% to 114.9% for all measured antimicrobials, and the within- and between-run precision values were <11.9% and <16.5% for the lower limit of quantification. No interferences and carry-over were observed. The samples were stable for at least 5 hours at room temperature or in the autosampler (10°C). CONCLUSIONS: The LC-MS/MS method developed in this study is appropriate and practical for the therapeutic drug monitoring of antimicrobials in the daily clinical laboratory practice because of its short analysis time, the need for a small amount of plasma, high specificity, and accuracy.

Cultivation-Free Raman Spectroscopic Investigations of Bacteria.

Raman spectroscopy is currently advertised as a hot and ambitious technology that has all of the features needed to characterize and identify bacteria. Raman spectroscopy is rapid, easy to use, noninvasive, and it could complement established microbiological and biomolecular methods in the near future. To bring this vision closer to reality, ongoing research is being conducted on spectral fingerprinting. This can yield a wealth of information, from even single bacteria from various habitats which can be further improved by combining Raman spectroscopy with methods such as stable isotope probing to elucidate microbial interactions. In conjunction with extensive statistical analysis, Raman spectroscopy will allow identification of (non)pathogenic bacteria at different taxonomic levels.