RESUMO
Bacteriophages, or "phages" for short, are viruses that replicate in bacteria. The therapeutic and biotechnological potential of phages and their lytic enzymes is of interest for their ability to selectively destroy pathogenic bacteria, including antibiotic-resistant strains. Introduction of phage preparations into medicine, biotechnology, and food industry requires a thorough characterization of phage-host interaction on a molecular level. We employed Raman tweezers to analyze the phage-host interaction of Staphylococcus aureus strain FS159 with a virulent phage JK2 (=812K1/420) of the Myoviridae family and a temperate phage 80α of the Siphoviridae family. We analyzed the timeline of phage-induced molecular changes in infected host cells. We reliably detected the presence of replicating phages in bacterial cells within 5 min after infection. Our results lay the foundations for building a Raman-based diagnostic instrument capable of real-time, in vivo, in situ, nondestructive characterization of the phage-host relationship on the level of individual cells, which has the potential of importantly contributing to the development of phage therapy and enzybiotics.
Assuntos
Bacteriófagos/química , Pinças Ópticas , Staphylococcus aureus/química , Análise Espectral RamanRESUMO
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.
Assuntos
Escherichia coli/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Pinças Ópticas , Antibacterianos/efeitos adversos , Escherichia coli/crescimento & desenvolvimento , Micromanipulação/métodos , Análise de Componente Principal , Análise Espectral RamanRESUMO
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.
Assuntos
Bactérias , Meios de Cultura/farmacologia , Análise Espectral Raman/métodos , Leveduras , Bactérias/química , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Leveduras/química , Leveduras/efeitos dos fármacos , Leveduras/metabolismoRESUMO
Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made.
Assuntos
Biofilmes , Candida/classificação , Análise Espectral Raman , Candida/citologia , Candida/ultraestruturaRESUMO
The feature issue of Biomedical Optics Express presents studies that were the focus of the Optical Manipulation and its Applications (OMA) meeting that was held on 24 - 27 April 2022 in Vancouver, Canada.
RESUMO
Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations-MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms' "fingerprint" (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.
Assuntos
Antibacterianos/farmacologia , Análise Espectral Raman/métodos , Ciprofloxacina/farmacologia , Clindamicina/farmacologia , DNA Bacteriano/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genéticaRESUMO
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.
Assuntos
Nanopartículas Metálicas , Análise Espectral Raman , Análise Espectral Raman/métodos , Nanopartículas Metálicas/química , Ouro/química , Anticorpos/química , Staphylococcus aureus , Escherichia coliRESUMO
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.
Assuntos
Sepse , Análise Espectral Raman , Humanos , Análise Espectral Raman/métodos , Projetos Piloto , Pinças Ópticas , AlgoritmosRESUMO
Pathogenic microbes contribute to several major global diseases that kill millions of people every year. Bloodstream infections caused by these microbes are associated with high morbidity and mortality rates, which are among the most common causes of hospitalizations. The search for the "Holy Grail" in clinical diagnostic microbiology, a reliable, accurate, low cost, real-time, and easy-to-use diagnostic method, is one of the essential issues in clinical practice. These very critical conditions can be met by Raman tweezers in combination with advanced analysis methods. Here, we present a proof-of-concept study based on Raman tweezers combined with spectral mixture analysis that allows for the identification of microbial strains directly from human blood serum without user intervention, thus eliminating the influence of a data analyst.
RESUMO
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.
Assuntos
Anti-Infecciosos , Análise Espectral Raman , Humanos , Análise de Célula Única , Análise Espectral Raman/métodos , Fatores de VirulênciaRESUMO
Urinary tract infections belong to the most common infections in the world. Besides community-acquired infections, nosocomial infections pose a high risk especially for patients having indwelling catheters, undergoing urological surgeries or staying at hospital for prolonged time. They can be often complicated by antimicrobial resistance and/or biofilm formation. Therefore, a rapid diagnostic tool enabling timely identification of a causative agent and its susceptibility to antimicrobials is a need. Raman spectroscopy appears to be a suitable method that allows rapid differentiation among microbes and provides a space for further analyses, such as determination of capability of biofilm formation or antimicrobial susceptibility/resistance in tested strains. Our work here presents a possibility to differ among most common microbes causing urinary tract infections (belonging to 20 species). We tested 254 strains directly from colonies grown on Mueller-Hinton agar plates. The results show that it is possible to distinguish among the tested species using Raman spectroscopy, which proves its great potential for future use in clinical diagnostics. Moreover, we present here a pilot study of a real-time analysis and identification (in less than 10 min) of single microbial cells directly in urine employing optical tweezers combined with Raman spectroscopy.
Assuntos
Análise Espectral Raman , Infecções Urinárias , Diferenciação Celular , Humanos , Projetos Piloto , Infecções Urinárias/diagnósticoRESUMO
Liquid crystal microdroplets with readily adjustable optical properties have attracted considerable attention for building reconfigurable optofluidic microsystems for sensing, imaging, and light routing applications. In this quest, development of active optical microcavities serving as versatile integrated sources of coherent light and ultra-sensitive environmental sensors has played a prominent role. Here, we study transportable optofluidic microlasers reversibly tunable by an external electric field, which are based on fluorophore-doped emulsion droplets of radial nematic liquid crystals manipulated by optical tweezers in microfluidic chips with embedded liquid electrodes. Full transparency of the electrodes formed by a concentrated electrolyte solution allows for applying an electric field to the optically trapped droplets without undesired heating caused by light absorption. Taking advantage of independent, precise control over the electric and thermal stimulation of the lasing liquid crystal droplets, we characterize their spectral tuning response at various optical trapping powers and study their relaxation upon a sudden decrease in the trapping power. Finally, we demonstrate that sufficiently strong applied electric fields can induce fully reversible phase transitions in the trapped droplets even below the bulk melting temperature of the used liquid crystal. Our observations indicate viability of creating electrically tunable, optically transported microlasers that can be prepared on-demand and operated within microfluidic chips to implement integrated microphotonic or sensing systems.
RESUMO
Selenium (Se) is an element with many commercial applications as well as an essential micronutrient. Dietary Se has antioxidant properties and it is known to play a role in cancer prevention. However, the general population often suffers from Se deficiency. Green algae, such as Chlorella vulgaris, cultivated in Se-enriched environment may be used as a food supplement to provide adequate levels of Se. We used Raman microspectroscopy (RS) for fast, reliable, and non-destructive measurement of Se concentration in living algal cells. We employed inductively coupled plasma-mass spectrometry as a reference method to RS and we found a substantial correlation between the Raman signal intensity at 252 cm-1 and total Se concentration in the studied cells. We used RS to assess the uptake of Se by living and inactivated algae and demonstrated the necessity of active cellular transport for Se accumulation. Additionally, we observed the intracellular Se being transformed into an insoluble elemental form, which we further supported by the energy-dispersive X-ray spectroscopy imaging.
Assuntos
Chlorella vulgaris/metabolismo , Selênio/metabolismo , Análise Espectral Raman , Bioacumulação , Chlorella vulgaris/química , Selênio/análise , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/metabolismoRESUMO
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 (120-360â¯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.
Assuntos
Celulose/análogos & derivados , Composição de Medicamentos/métodos , Implantes de Medicamento/química , Glucose/química , Polietilenoglicóis/química , Amido/análogos & derivados , Celulose/química , Química Farmacêutica , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Excipientes/química , Glucose/farmacologia , Temperatura Alta , Hipoglicemia/prevenção & controle , Tamanho da Partícula , Solubilidade , Amido/química , Propriedades de SuperfícieRESUMO
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.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida parapsilosis/metabolismo , Análise Espectral Raman/métodos , Staphylococcus epidermidis/metabolismo , Testes Diagnósticos de Rotina/métodos , HumanosRESUMO
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.
Assuntos
Análise Espectral Raman/métodos , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Técnicas Bacteriológicas , Contagem de Colônia Microbiana/métodos , República Tcheca , Humanos , Staphylococcus aureus/patogenicidade , Staphylococcus epidermidis/patogenicidadeRESUMO
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.
Assuntos
Análise Espectral Raman/métodos , Staphylococcus/isolamento & purificação , Ágar , Testes Diagnósticos de Rotina , Fluorescência , Análise de Componente Principal , Manejo de Espécimes , Fatores de TempoRESUMO
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.