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.

• Publication type
• Journal Article MeSH

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.

• Keywords
• Staphylococcus capitis, bacterial pigments, carotenoids, coagulase-negative staphylococci (CoNS), staphyloxanthin,
• Publication type
• Journal Article MeSH

Melanins are complex pigments with various biological functions and potential applications in space exploration and biomedicine due to their radioprotective properties. Aspergillus niger, a fungus known for its high radiation resistance, is widely used in biotechnology and a candidate for melanin production. In this study, we investigated the production of fungal pyomelanin (PyoFun) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant ΔhmgA mutant strain (OS4.3). The PyoFun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of PyoFun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (PyoSyn). The results confirmed successful production of PyoFun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of PyoFun, and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, PyoFun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by PyoSyn. The findings of this study suggest that PyoFun has significant potential as a biological shield against harmful radiation. Notably, PyoFun is synthesized extracellularly, differing it from other fungal melanins (such as L-DOPA- or DHN-melanin) that require cell lysis for pigment purification. This characteristic makes PyoFun a valuable resource for biotechnology, biomedicine, and the space industry. However, further research is needed to evaluate its protective effect in a dried form and against ionizing radiation.

• Keywords
• Aspergillus niger, cosmic radiation, melanin, pyomelanin, radioprotection, space exploration,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Candida, Raman spectroscopy, Raman tweezers, bacteria, bloodstream infections, diagnostics, sepsis,
• MeSH
• Algorithms MeSH
• Humans MeSH
• Optical Tweezers MeSH
• Pilot Projects MeSH
• Spectrum Analysis, Raman * methods   MeSH
• Sepsis * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• Keywords
• Escherichia coli, SERS-tag, sandwich immunoassay, single-cell detection,
• MeSH
• Escherichia coli MeSH
• Metal Nanoparticles * chemistry   MeSH
• Antibodies chemistry   MeSH
• Spectrum Analysis, Raman * methods   MeSH
• Staphylococcus aureus MeSH
• Gold chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antibodies MeSH
• Gold MeSH

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.

• Keywords
• Raman spectroscopy, biofilm, cryo-SEM, sample preparation, scanning electron microscopy,
• MeSH
• Bacterial Infections diagnosis   microbiology   MeSH
• Biofilms growth & development   MeSH
• Candida parapsilosis isolation & purification   pathogenicity   ultrastructure   MeSH
• Humans MeSH
• Microscopy, Electron, Scanning MeSH
• Spectrum Analysis, Raman MeSH
• Staphylococcus epidermidis isolation & purification   pathogenicity   ultrastructure   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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.

• MeSH
• Agar MeSH
• Principal Component Analysis MeSH
• Time Factors MeSH
• Diagnostic Tests, Routine MeSH
• Fluorescence MeSH
• Specimen Handling MeSH
• Spectrum Analysis, Raman methods   MeSH
• Staphylococcus isolation & purification   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Agar MeSH