The search for the "Holy Grail" in clinical diagnostic microbiology-a reliable, accurate, low-cost, real-time, easy-to-use method-has brought up several methods with the potential to meet these criteria. One is Raman spectroscopy, an optical, nondestructive method based on the inelastic scattering of monochromatic light. The current study focuses on the possible use of Raman spectroscopy for identifying microbes causing severe, often life-threatening bloodstream infections. We included 305 microbial strains of 28 species acting as causative agents of bloodstream infections. Raman spectroscopy identified the strains from grown colonies, with 2.8% and 7% incorrectly identified strains using the support vector machine algorithm based on centered and uncentred principal-component analyses, respectively. We combined Raman spectroscopy with optical tweezers to speed up the process and captured and analyzed microbes directly from spiked human serum. The pilot study suggests that it is possible to capture individual microbial cells from human serum and characterize them by Raman spectroscopy with notable differences among different species. IMPORTANCE Bloodstream infections are among the most common causes of hospitalizations and are often life-threatening. To establish an effective therapy for a patient, the timely identification of the causative agent and characterization of its antimicrobial susceptibility and resistance profiles are essential. Therefore, our multidisciplinary team of microbiologists and physicists presents a method that reliably, rapidly, and inexpensively identifies pathogens causing bloodstream infections-Raman spectroscopy. We believe that it might become a valuable diagnostic tool in the future. Combined with optical trapping, it offers a new approach where the microorganisms are individually trapped in a noncontact way by optical tweezers and investigated by Raman spectroscopy directly in a liquid sample. Together with the automatic processing of measured Raman spectra and comparison with a database of microorganisms, it makes the whole identification process almost real time.
- MeSH
- algoritmy MeSH
- lidé MeSH
- optická pinzeta MeSH
- pilotní projekty MeSH
- Ramanova spektroskopie * metody MeSH
- sepse * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.
Rapid and accurate identification of pathogens causing infections is one of the biggest challenges in medicine. Timely identification of causative agents and their antimicrobial resistance profile can significantly improve the management of infection, lower costs for healthcare, mitigate ever-growing antimicrobial resistance and in many cases, save lives. Raman spectroscopy was shown to be a useful-quick, non-invasive, and non-destructive -tool for identifying microbes from solid and liquid media. Modifications of Raman spectroscopy and/or pretreatment of samples allow single-cell analyses and identification of microbes from various samples. It was shown that those non-culture-based approaches could also detect antimicrobial resistance. Moreover, recent studies suggest that a combination of Raman spectroscopy with optical tweezers has the potential to identify microbes directly from human body fluids. This review aims to summarize recent advances in non-culture-based approaches of identification of microbes and their virulence factors, including antimicrobial resistance, using methods based on Raman spectroscopy in the context of possible use in the future point-of-care diagnostic process.
- MeSH
- analýza jednotlivých buněk MeSH
- antiinfekční látky * MeSH
- faktory virulence MeSH
- lidé MeSH
- Ramanova spektroskopie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Aim: Finding rapid, reliable diagnostic methods is a big challenge in clinical microbiology. Raman spectroscopy is an optical method used for multiple applications in scientific fields including microbiology. This work reports its potential in identifying biofilm positive strains of Candida parapsilosis and Staphylococcus epidermidis. Materials & methods: We tested 54 S. epidermidis strains (23 biofilm positive, 31 negative) and 51 C. parapsilosis strains (27 biofilm positive, 24 negative) from colonies on Mueller-Hinton agar plates, using Raman spectroscopy. Results: The accuracy was 98.9% for C. parapsilosis and 96.1% for S. epidermidis. Conclusion: The method showed great potential for identifying biofilm positive bacterial and yeast strains. We suggest that Raman spectroscopy might become a useful aid in clinical diagnostics.
Due to the additional particle coalescence in the coating, changes in the dissolution profile occur over time in the formulations coated by aqueous ethylcellulose latex. Dry thermal treatment (DT) of the coating can be used as a prevention of this process. Alternatively, it is advisable to take advantage of the synergistic effect of high humidity during wet treatment (WT), which substantially accelerates the film formation. This can be a problem for time-controlled systems, which are based on the coating rupture due to the penetration of water into the core causing the increase in the system volume. This process can begin already during the WT, which may affect the coating adversely. The submitted work was focused on the stability testing of two pellet core compositions: pellets containing swelling superdisintegrant sodium carboxymethyl starch (CMS) and pellets containing osmotically active polyethylene glycol (PEG). Another objective was to identify the treatment/storage condition effects on the pellet dissolution profiles. These pellets are intended to prevent hypoglycemia for patients with diabetes mellitus and therefore, besides the excipients, pellet cores contain 75% or 80% of glucose. The pellet coating is formed by ethylcellulose-based latex, which provides the required lag time (120-360 min). The sample stability was evaluated depending on the pellet core composition (PEG, CMS) for two types of final pellet coating treatment (DT or WT). Scanning electron microscopy and Raman microspectroscopy revealed the penetration of glucose and polyethylene glycol from the core to the PEG pellet surface after WT. For the CMS sample, significant pellet swelling after WT (under the conditions of elevated humidity) was statistically confirmed by the means of stereomicroscopic data evaluation. Therefore, the acceleration of dissolution rate during the stress tests is caused by the soluble substance penetration through the coating in the case of PEG pellets or by dosage form volume increase in the case of CMS pellets. The observed mechanisms can be generally anticipated during the stability testing of the ethylcellulose coated dosage forms. The aforementioned processes do not occur after DT and the pellets are stable in the environment without increased humidity.
- MeSH
- celulosa analogy a deriváty chemie MeSH
- chemie farmaceutická MeSH
- glukosa chemie farmakologie MeSH
- hypoglykemie prevence a kontrola MeSH
- léky implantované chemie MeSH
- léky s prodlouženým účinkem MeSH
- polyethylenglykoly chemie MeSH
- pomocné látky chemie MeSH
- povrchové vlastnosti MeSH
- příprava léků metody MeSH
- rozpustnost MeSH
- škrob analogy a deriváty chemie MeSH
- stabilita léku MeSH
- uvolňování léčiv MeSH
- velikost částic MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
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.
Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.
AIM: Raman spectroscopy is an analytical method with a broad range of applications across multiple scientific fields. We report on a possibility to differentiate between two important Gram-positive species commonly found in clinical material - Staphylococcus aureus and Staphylococcus epidermidis - using this rapid noninvasive technique. MATERIALS & METHODS: For this, we tested 87 strains, 41 of S. aureus and 46 of S. epidermidis, directly from colonies grown on a Mueller-Hinton agar plate using Raman spectroscopy. DISCUSSION & CONCLUSION: The method paves a way for separation of these two species even on high number of samples and therefore, it can be potentially used in clinical diagnostics.
- MeSH
- bakteriologické techniky MeSH
- lidé MeSH
- počet mikrobiálních kolonií metody MeSH
- Ramanova spektroskopie metody MeSH
- stafylokokové infekce diagnóza MeSH
- Staphylococcus aureus izolace a purifikace patogenita MeSH
- Staphylococcus epidermidis izolace a purifikace patogenita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Raman spectroscopy has a broad range of applications across numerous scientific fields, including microbiology. Our work here monitors the influence of culture media on the Raman spectra of clinically important microorganisms (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans). Choosing an adequate medium may enhance the reproducibility of the method as well as simplifying the data processing and the evaluation. We tested four different media per organism depending on the nutritional requirements and clinical usage directly on a Petri dish. Some of the media have a significant influence on the microbial fingerprint (Roosvelt-Park Institute Medium, CHROMagar) and should not be used for the acquisition of Raman spectra. It was found that the most suitable medium for microbiological experiments regarding these organisms was Mueller-Hinton agar.
A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.
- MeSH
- algoritmy MeSH
- analýza hlavních komponent MeSH
- Bacteria metabolismus MeSH
- biofilmy * MeSH
- buněčná adheze MeSH
- fagocytóza MeSH
- fyziologie bakterií MeSH
- optická pinzeta * MeSH
- polysacharidy chemie MeSH
- Ramanova spektroskopie * MeSH
- Staphylococcus epidermidis metabolismus MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
