Identification of staphyloxanthin and derivates in yellow-pigmented Staphylococcus capitis subsp. capitis.

Introduction: Staphylococcus capitis naturally colonizes the human skin but as an opportunistic pathogen, it can also cause biofilm-associated infections and bloodstream infections in newborns. Previously, we found that two strains from the subspecies S. capitis subsp. capitis produce yellow carotenoids despite the initial species description, reporting this subspecies as non-pigmented. In Staphylococcus aureus, the golden pigment staphyloxanthin is an important virulence factor, protecting cells against reactive oxygen species and modulating membrane fluidity. Methods: In this study, we used two pigmented (DSM 111179 and DSM 113836) and two non-pigmented S. capitis subsp. capitis strains (DSM 20326T and DSM 31028) to identify the pigment, determine conditions under which pigment-production occurs and investigate whether pigmented strains show increased resistance to ROS and temperature stress. Results: We found that the non-pigmented strains remained colorless regardless of the type of medium, whereas intensity of pigmentation in the two pigmented strains increased under low nutrient conditions and with longer incubation times. We were able to detect and identify staphyloxanthin and its derivates in the two pigmented strains but found that methanol cell extracts from all four strains showed ROS scavenging activity regardless of staphyloxanthin production. Increased survival to cold temperatures (-20°C) was detected in the two pigmented strains only after long-term storage compared to the non-pigmented strains. Conclusion: The identification of staphyloxanthin in S. capitis is of clinical relevance and could be used, in the same way as in S. aureus, as a possible target for anti-virulence drug design.

Physiological and transcriptome profiling of Chlorella sorokiniana: A study on azo dye wastewater decolorization.

Over decades, synthetic dyes have become increasingly dominated by azo dyes posing a significant environmental risk due to their toxicity. Microalgae-based systems may offer an alternative for treatment of azo dye effluents to conventional physical-chemical methods. Here, microalgae were tested to decolorize industrial azo dye wastewater (ADW). Chlorella sorokiniana showed the highest decolorization efficiency in a preliminary screening test. Subsequently, the optimization of the experimental design resulted in 70% decolorization in a photobioreactor. Tolerance of this strain was evidenced using multiple approaches (growth and chlorophyll content assays, scanning electron microscopy (SEM), and antioxidant level measurements). Raman microspectroscopy was employed for the quantification of ADW-specific compounds accumulated by the microalgal biomass. Finally, RNA-seq revealed the transcriptome profile of C. sorokiniana exposed to ADW for 72 h. Activated DNA repair and primary metabolism provided sufficient energy for microalgal growth to overcome the adverse toxic conditions. Furthermore, several transporter genes, oxidoreductases-, and glycosyltransferases-encoding genes were upregulated to effectively sequestrate and detoxify the ADW. This work demonstrates the potential utilization of C. sorokiniana as a tolerant strain for industrial wastewater treatment, emphasizing the regulation of its molecular mechanisms to cope with unfavorable growth conditions.

Rapid Identification of Pathogens Causing Bloodstream Infections by Raman Spectroscopy and Raman Tweezers.

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.

SERS-Tags: Selective Immobilization and Detection of Bacteria by Strain-Specific Antibodies and Surface-Enhanced Raman Scattering.

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.

Laser-based techniques: Novel tools for the identification and characterization of aged microplastics with developed biofilm.

Microplastics found in the environment are often covered with a biofilm, which makes their analysis difficult. Therefore, the biofilm is usually removed before analysis, which may affect the microplastic particles or lead to their loss during the procedure. In this work, we used laser-based analytical techniques and evaluated their performance in detecting, characterizing, and classifying pristine and aged microplastics with a developed biofilm. Five types of microplastics from different polymers were selected (polyamide, polyethylene, polyethylene terephthalate, polypropylene, and polyvinyl chloride) and aged under controlled conditions in freshwater and wastewater. The development of biofilm and the changes in the properties of the microplastic were evaluated. The pristine and aged microplastics were characterized by standard methods (e.g., optical and scanning electron microscopy, and Raman spectroscopy), and then laser-induced breakdown spectroscopy (LIBS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) were used. The results show that LIBS could identify different types of plastics regardless of the ageing and major biotic elements of the biofilm layer. LA-ICP-MS showed a high sensitivity to metals, which can be used as markers for various plastics. In addition, LA-ICP-MS can be employed in studies to monitor the adsorption and desorption (leaching) of metals during the ageing of microplastics. The use of these laser-based analytical techniques was found to be beneficial in the study of environmentally relevant microplastics.

Rapid identification of pathogens in blood serum via Raman tweezers in combination with advanced processing methods.

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.

Raman Spectroscopy-A Novel Method for Identification and Characterization of Microbes on a Single-Cell Level in Clinical Settings.

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.

The role of polyhydroxyalkanoates in adaptation of Cupriavidus necator to osmotic pressure and high concentration of copper ions.

Polyhydroxyalkanoates (PHA) are abundant microbial polyesters accumulated in the form of intracellular granules by numerous prokaryotes primarily as storage of carbon and energy. Apart from their storage function, the presence of PHA also enhances the robustness of the microbial cells against various stressors. In this work, we investigated the role of PHA in Cupriavidus necator, a model organism concerning PHA metabolism, for adaptation to osmotic pressure and copper ions. In long-term laboratory evolution experiments, the bacterial culture was cultivated in presence of elevated doses of sodium chloride or copper ions (incubations lasted 78 passages for Cu2+ and 68 passages for NaCl) and the evolved strains were compared with the wild-type strain in terms of growth and PHA production capacity, cell morphology (investigated by various electron microscopy techniques), activities of selected enzymes involved in PHA metabolism and other crucial metabolic pathways, the chemical composition of bacterial biomass (determined by infrared and Raman spectroscopy) and also considering robustness against various stressors. The results confirmed the important role of PHA metabolism for adaptation to both tested stressors.

Raman spectroscopy-a tool for rapid differentiation among microbes causing urinary tract infections.

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.

Use of Waste Substrates for the Lipid Production by Yeasts of the Genus Metschnikowia-Screening Study.

Oleogenic yeasts are characterized by the ability to accumulate increased amounts of lipids under certain conditions. These microbial lipids differ in their fatty acid composition, which allows them to be widely used in the biotechnology industry. The interest of biotechnologists is closely linked to the rising prices of fossil fuels in recent years. Their negative environmental impact is caused by significantly increased demand for biodiesel. The composition of microbial lipids is very similar to vegetable oils, which provides great potential for use in the production of biodiesel. In addition, some oleogenic microorganisms are capable of producing lipids with a high proportion of unsaturated fatty acids. The presented paper's main aim was to study the production of lipids and lipid substances by yeasts of the genus Metschnikowia, to cultivate crude waste animal fat to study its utilization by yeasts, and to apply the idea of circular economy in the biotechnology of Metschnikowia yeasts. The work focuses on the influence of various stress factors in the cultivation process, such as reduced temperature or nutritional stress through the use of various waste substrates, together with manipulating the ratio of carbon and nitrogen sources in the medium. Yeast production properties were monitored by several instrumental techniques, including gas chromatography and Raman spectroscopy. The amount of lipids and in particular the fatty acid composition varied depending on the strains studied and the culture conditions used. The ability of yeast to produce significant amounts of unsaturated fatty acids was also demonstrated in the work. The most suitable substrate for lipid production was a medium containing glycerol, where the amount of accumulated lipids in the yeast M. pulcherrima 1232 was up to 36%. In our work, the crude animal fat was used for the production of high-value lipids, which to the best of our knowledge is a new result. Moreover, quantitative screening of lipase enzyme activity cultivated on animal fat substrate on selected yeasts of the genus Metschnikowia was performed. We found that for the yeast utilizing glycerol, animal fat seems to be an excellent source of carbon. Therefore, the yeast conversion of crude processed animal fat to value-added products is a valuable process for the biotechnology and food industry.

Raman Microspectroscopic Analysis of Selenium Bioaccumulation by Green Alga Chlorella vulgaris.

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.

The Use of Raman Spectroscopy to Monitor Metabolic Changes in Stressed Metschnikowia sp. Yeasts.

Raman spectroscopy is a universal method designed for the analysis of a wide range of physical, chemical and biological systems or various surfaces. This technique is suitable to monitor various components of cells, tissues or microorganisms. The advantages include very fast non-contact and non-destructive analysis and no or minimal need for sample treatment. The yeasts Metschnikowia can be considered as industrially usable producers of pulcherrimin or single-cell lipids, depending on cultivation conditions and external stress. In the present study, Raman spectroscopy was used as an effective tool to identify both pulcherrimin and lipids in single yeast cells. The analysis of pulcherrimin is very demanding; so far, there is no optimal procedure to analyze or identify this pigment. Based on results, the strong dependence of pulcherrimin production on the ferric ion concentration was found with the highest yield in media containing 0.1 g/L iron. Further, production of lipids in Metschnikowia cells was studied at different temperatures and C:N ratios, using Raman spectroscopy to follow fatty acids composition, under different regimes, by monitoring the iodine number. The results of Raman spectroscopy were comparable with the fatty acid analysis obtained by gas chromatography. This study therefore supported use of Raman spectroscopy for biotechnological applications as a simple tool in the identification and analysis both the pulcherrimin and microbial lipids. This method provides a quick and relatively accurate estimation of targeted metabolites with minimal sample modification and allows to monitor metabolic changes over time of cultivation.

Analysis of Bacteriophage-Host Interaction by Raman Tweezers.

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.

Identification of ability to form biofilm in Candida parapsilosis and Staphylococcus epidermidis by Raman spectroscopy.

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.

The effects of the treatment conditions on the dissolution profile of ethylcellulose coated pellets.

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.

What keeps polyhydroxyalkanoates in bacterial cells amorphous? A derivation from stress exposure experiments.

Polyhydroxyalkanoates (PHA) are storage polymers accumulated by numerous prokaryotes in form of intracellular granules. Native PHA granules are formed by amorphous polymer which reveals considerably higher elasticity and flexibility as compared to crystalline pure PHA polymers. The fact that bacteria store PHA in amorphous state has great biological consequences. It is not clear which mechanisms protect amorphous polymer in native granules from transition into thermodynamically favorable crystalline state. Here, we demonstrate that exposition of bacterial cells to particular stressors induces granules aggregation, which is the first but not sufficient condition for PHA crystallization. Crystallization of the polymer occurs only when the stressed bacterial cells are subsequently dried. The fact that both granules aggregation and cell drying must occur to induce crystallization of PHA indicates that both previously suggested hypotheses about mechanisms of stabilization of amorphous state of native PHA are valid and, in fact, both effects participate synergistically. It seems that the amorphous state of the polymer is stabilized kinetically by the low rate of crystallization in limited volume in small PHA granules and, moreover, water present in PHA granules seems to function as plasticizer protecting the polymer from crystallization, as confirmed experimentally for the first time by the present work.

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy.

The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

Detection of Chloroalkanes by Surface-Enhanced Raman Spectroscopy in Microfluidic Chips.

Optofluidics, a research discipline combining optics with microfluidics, currently aspires to revolutionize the analysis of biological and chemical samples, e.g., for medicine, pharmacology, or molecular biology. In order to detect low concentrations of analytes in water, we have developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The geometry of the gold surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system, which allowed transport and precise mixing of the analyzed fluids, while preventing contamination or abrasion of the highly sensitive substrate. To illustrate its practical use, we employed the device for quantitative detection of persistent environmental pollutant 1,2,3-trichloropropane in water in submillimolar concentrations. The developed sensor allows fast and simple quantification of halogenated compounds and it will contribute towards the environmental monitoring and enzymology experiments with engineered haloalkane dehalogenase enzymes.

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.