Metabolic response of Klebsiella oxytoca to ciprofloxacin exposure: a metabolomics approach.

Introduction: Rapid detection and identification of pathogens and antimicrobial susceptibility is essential for guiding appropriate antimicrobial therapy and reducing morbidity and mortality associated with sepsis.

Objectives: The metabolic response of clinical isolates of Klebsiella oxytoca exposed to different concentrations of ciprofloxacin (the second generation of quinolones antibiotics) were studied in order to investigate underlying mechanisms associated with antimicrobial resistance (AMR).

Methods: Metabolomics investigations were performed using Fourier-transform infrared (FT-IR) spectroscopy as a metabolic fingerprinting approach combined with gas chromatography-mass spectrometry (GC-MS) for metabolic profiling.

Application of infrared spectroscopy to study carbon-deuterium kinetics and isotopic spectral shifts at the single-cell level.

Microbial communities play crucial roles in shaping natural ecosystems, impacting human well-being, and driving advancements in industrial biotechnology. However, associating specific metabolic functions with bacteria proves challenging due to the vast diversity of microorganisms within these communities. In the past decades stable isotope probing (SIP) approaches, coupled with vibrational spectroscopy, have emerged as a novel method for revealing microbial metabolic roles and interactions in complex communities.

Rapid Classification and Differentiation of Sepsis-Related Pathogens Using FT-IR Spectroscopy.

Sepsis is a life-threatening condition arising from a dysregulated host immune response to infection, leading to a substantial global health burden. The accurate identification of bacterial pathogens in sepsis is essential for guiding effective antimicrobial therapy and optimising patient outcomes. Traditional culture-based bacterial typing methods present inherent limitations, necessitating the exploration of alternative diagnostic approaches.

Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. 104 pathogenic isolates of , , and were analyzed. Overall prediction accuracy was 99.

Monitoring Phenotype Heterogeneity at the Single-Cell Level within Populations Producing Poly-3-hydroxybutyrate by Label-Free Super-resolution Infrared Imaging.

Phenotypic heterogeneity is commonly found among bacterial cells within microbial populations due to intrinsic factors as well as equipping the organisms to respond to external perturbations. The emergence of phenotypic heterogeneity in bacterial populations, particularly in the context of using these bacteria as microbial cell factories, is a major concern for industrial bioprocessing applications. This is due to the potential impact on overall productivity by allowing the growth of subpopulations consisting of inefficient producer cells.

Metabolomics of for Disclosing Novel Metabolic Engineering Strategies for Enhancing Hydrogen and Ethanol Production.

The biological production of hydrogen is an appealing approach to mitigating the environmental problems caused by the diminishing supply of fossil fuels and the need for greener energy. is one of the best-characterized microorganisms capable of consuming glycerol-a waste product of the biodiesel industry-and producing H and ethanol. However, the natural capacity of to generate these compounds is insufficient for commercial or industrial purposes.

Unlocking the secrets of the microbiome: exploring the dynamic microbial interplay with humans through metabolomics and their manipulation for synthetic biology applications.

Metabolomics is a powerful research discovery tool with the potential to measure hundreds to low thousands of metabolites. In this review, we discuss the application of GC-MS and LC-MS in discovery-based metabolomics research, we define metabolomics workflows and we highlight considerations that need to be addressed in order to generate robust and reproducible data. We stress that metabolomics is now routinely applied across the biological sciences to study microbiomes from relatively simple microbial systems to their complex interactions within consortia in the host and the environment and highlight this in a range of biological species and mammalian systems including humans.

Optical photothermal infrared spectroscopy: A novel solution for rapid identification of antimicrobial resistance at the single-cell level deuterium isotope labeling.

The rise and extensive spread of antimicrobial resistance (AMR) has become a growing concern, and a threat to the environment and human health globally. The majority of current AMR identification methods used in clinical setting are based on traditional microbiology culture-dependent techniques which are time-consuming or expensive to be implemented, thus appropriate antibiotic stewardship is provided retrospectively which means the first line of treatment is to hope that a broad-spectrum antibiotic works. Hence, culture-independent and single-cell technologies are needed to allow for rapid detection and identification of antimicrobial-resistant bacteria and to support a more targeted and effective antibiotic therapy preventing further development and spread of AMR.

Simultaneous Raman and infrared spectroscopy: a novel combination for studying bacterial infections at the single cell level.

Sepsis is a life-threatening clinical condition responsible for approximately 11 million deaths worldwide. Rapid and accurate identification of pathogenic bacteria and its antimicrobial susceptibility play a critical role in reducing the morbidity and mortality rates related to sepsis. Raman and infrared spectroscopies have great potential to be used as diagnostic tools for rapid and culture-free detection of bacterial infections.

Metabolomics for the design of new metabolic engineering strategies for improving aerobic succinic acid production in Escherichia coli.

Introduction: Glycerol is a byproduct from the biodiesel industry that can be biotransformed by Escherichia coli to high added-value products such as succinate under aerobic conditions. The main genetic engineering strategies to achieve this aim involve the mutation of succinate dehydrogenase (sdhA) gene and also those responsible for acetate synthesis including acetate kinase, phosphate acetyl transferase and pyruvate oxidase encoded by ackA, pta and pox genes respectively in the ΔsdhAΔack-ptaΔpox (M4) mutant. Other genetic manipulations to rewire the metabolism toward succinate consist on the activation of the glyoxylate shunt or blockage the pentose phosphate pathway (PPP) by deletion of isocitrate lyase repressor (iclR) or gluconate dehydrogenase (gnd) genes on M4-ΔiclR and M4-Δgnd mutants respectively.

Metabolic Fingerprint Analysis of Cytochrome -producing N4830-1 Using FT-IR Spectroscopy.

Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome (CYT ) production in N4830-1, as the heterologous host. Cyt b was introduced as a plasmid with between 0 and 6 copies under a strong promoter.

Simultaneous Raman and Infrared Spectroscopy of Stable Isotope Labelled .

We report the use of a novel technology based on optical photothermal infrared (O-PTIR) spectroscopy for obtaining simultaneous infrared and Raman spectra from the same location of the sample allowing us to study bacterial metabolism by monitoring the incorporation of C- and N-labeled compounds. Infrared data obtained from bulk populations and single cells via O-PTIR spectroscopy were compared to conventional Fourier transform infrared (FTIR) spectroscopy in order to evaluate the reproducibility of the results achieved by all three approaches. Raman spectra acquired were concomitant with infrared data from bulk populations as well as infrared spectra collected from single cells, and were subjected to principal component analysis in order to evaluate any specific separation resulting from the isotopic incorporation.

The Role of Raman Spectroscopy Within Quantitative Metabolomics.

Ninety-four years have passed since the discovery of the Raman effect, and there are currently more than 25 different types of Raman-based techniques. The past two decades have witnessed the blossoming of Raman spectroscopy as a powerful physicochemical technique with broad applications within the life sciences. In this review, we critique the use of Raman spectroscopy as a tool for quantitative metabolomics.

Imaging Isotopically Labeled Bacteria at the Single-Cell Level Using High-Resolution Optical Infrared Photothermal Spectroscopy.

We report that the cellular uptake of stable isotope-labeled compounds by bacteria can be probed at the single-cell level using infrared spectroscopy, and this monitors the chemical vibrations affected by the incorporation of "heavy" atoms by cells and thus can be used to understand microbial systems. This presents a significant advancement as most studies have focused on evaluating communities of cells due to the poor spatial resolution achieved by classical infrared microspectrometers, and to date, there is no study evaluating the incorporation of labeled compounds by bacteria at single-cell levels using infrared spectroscopy. The development of new technologies and instrumentations that provide information on the metabolic activity of a single bacterium is critical as this will allow for a better understanding of the interactions between microorganisms as well as the function of individual members and their interactions in different microbial communities.

Metabolism in action: stable isotope probing using vibrational spectroscopy and SIMS reveals kinetic and metabolic flux of key substrates.

Microbial communities play essential functions which drive various ecosystems supporting animal and aquatic life. However, linking bacteria with specific metabolic functions is difficult, since microbial communities consist of numerous and phylogenetically diverse microbes. Stable isotope probing (SIP) combined with single-cell tools has emerged as a novel culture-independent strategy for unravelling microbial metabolic roles and intertwined interactions in complex communities.

Discrimination of bacteria using whole organism fingerprinting: the utility of modern physicochemical techniques for bacterial typing.

Rapid and accurate classification and discrimination of bacteria is an important task and has been highlighted recently for rapid diagnostics using real-time results. Coupled with a recent report by Jim O'Neill [] that if left unaddressed antimicrobial resistance (AMR) in bacteria could kill 10 million people per year by 2050, which would surpass current cancer mortality, this further highlights the need for unequivocal identification of microorganisms. Whilst traditional microbiological testing has offered insights into the characterisation and identification of a wide range of bacteria, these approaches have proven to be laborious and time-consuming and are not really fit for purpose, considering the modern day speed and volume of international travel and the opportunities it creates for the spread of pathogens globally.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable.

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence.

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine.

Evaluation of Sample Preparation Methods for Inter-Laboratory Metabolomics Investigation of TK24.

In the past two decades, metabolomics has proved to be a valuable tool with many potential applications in different areas of science. However, there are still some challenges that need to be addressed, particularly for multicenter studies. These challenges are mainly attributed to various sources of fluctuation and unwanted variations that can be introduced at pre-analytical, analytical, and/or post-analytical steps of any metabolomics experiment.

Assessment of Transdermal Delivery of Topical Compounds in Skin Scarring Using a Novel Combined Approach of Raman Spectroscopy and High-Performance Liquid Chromatography.

The goal of any topical formulation is efficient transdermal delivery of its active components. However, delivery of compounds can be problematic with penetration through tough layers of fibrotic dermal scar tissue. We propose a new approach combining high-performance liquid chromatography (HPLC) and Raman spectroscopy (RS) using a topical of unknown composition against a well-known antiscar topical (as control).

Radiation Tolerance of , a Cyanobacterium Relevant to the First Generation Magnox Storage Pond.

Recently a species of was identified as the dominant photosynthetic organism during a bloom event in a high pH (pH ∼11.4), radioactive spent nuclear fuel pond (SNFP) at the Sellafield Ltd., United Kingdom facility.

A microbiome and metabolomic signature of phases of cutaneous healing identified by profiling sequential acute wounds of human skin: An exploratory study.

Profiling skin microbiome and metabolome has been utilised to gain further insight into wound healing processes. The aims of this multi-part temporal study in 11 volunteers were to analytically profile the dynamic wound tissue and headspace metabolome and sequence microbial communities in acute wound healing at days 0, 7, 14, 21 and 28, and to investigate their relationship to wound healing, using non-invasive quantitative devices. Metabolites were obtained using tissue extraction, sorbent and polydimethylsiloxane patches and analysed using GCMS.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines.

Rapid differentiation of Campylobacter jejuni cell wall mutants using Raman spectroscopy, SERS and mass spectrometry combined with chemometrics.

The Gram-negative bacterial pathogen Campylobacter jejuni is a major cause of foodborne gastroenteritis worldwide. Rapid detection and identification of C. jejuni informs timely prescription of appropriate therapeutics and epidemiological investigations.